Therapeutic pyrazolyl thienopyridines

ABSTRACT

The present invention provides for compounds of Formula I, and pharmaceutically acceptable salts thereof, 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7  have any of the values defined therefor in the specification, and pharmaceutically acceptable salts thereof, that are useful as therapeutic agents in the treatment of TGFβ-mediated conditions, including cancer and fibrotic disorders. Also provided are pharmaceutical compositions comprising one or more compounds of Formula I.

BACKGROUND OF THE INVENTION

TGF-β's (transforming growth factor-β's) activate fibrotic andtumor-promoting signaling cascades. Three mammalian TGFβ's, TGFβ₁,TGFβ₂, and TGFβ₃, can activate the TGFβ pathway. The TGFβ's bind to andsignal through cell surface receptors (see Singh et al. (2004) Curr.Opin. Drug Disc. and Dev., 7: 437-445). A TGFβ first binds to a type IIreceptor (TβRII), which then binds to and phosphorylates a type Ireceptor (TβRI) (i.e., an activin receptor-like kinase (ALK)). There isa family of ALK proteins including ALK-5, which is the most specific ALKfor TGFβ. Activation of ALK-5 leads to phosporylation of intracellularproteins, which results in the regulation of fibrosis and tumorigenesis.Therefore, the discovery of ALK-5 inhibitors is an active area ofinvestigation to discover inhibitors to treat cancer, and conditionsinvolving fibrosis (see Singh et al. (2004)).

One example of a condition that involves fibrosis is the formation ofscars during wound repair. Scars, including hypertrophic and keloidscars, typically result from the deposition of collagen at wound sites.Wounds may be produced through many different kinds of mechanismsincluding surgery, accidental injuries, burns, trauma, etc. It has beenreported that the application of TGFβ₃, antibodies to TGFβ₁ and TGFβ₂which inhibit the TGFβ pathway can assist in reducing scarring (O'Kaneand Ferguson, (1997) Int. J. Biochem. Cell Biol., 29: 63-78).Accordingly, there is an ongoing need in the art for small moleculeALK-5 inhibitors that can be used to reduce scar formation, and for thetreatment of other fibrotic conditions, as well as cancer.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides for compounds of formulaI:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is athieno[3,2-c]pyridinyl, a thieno[3,2-b]pyridinyl, athieno[2,3-c]pyridinyl, or a thieno[2,3-b]pyridinyl, each of which maybe optionally substituted with one to three substituents eachindependently selected from the group consisting of: —C₁-C₃-alkyl,—(C₁-C₃-alkyl)-S—(C₁-C₃-alkyl), —S—C₁-C₃-alkyl,—(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl, —C(O)O—C₁-C₃-alkyl,—C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, wherein R³⁰ and R³¹ are eachindependently selected from the group consisting of: H, and —C₁-C₃alkyl-OH, —C₁-C₃-alkyl, halo, and —O—C₁-C₃-alkyl; R² and R³ areindependently selected from the group consisting of: hydrogen,—C₁-C₃-alkyl, —(C₁-C₃-alkyl)-S—(C₁-C₃-alkyl), —S—C₁-C₃-alkyl,—(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl, —C(O)O—C₁-C₃-alkyl,—C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, and a C₃-C₆-cycloalkyl, whereinR³⁰ and R³¹ are each independently selected from the group consistingof: H, and —C₁-C₃ alkyl; or R² and R³ may be taken together to form a 5or 6-membered heteroaryl, a phenyl, a C₄-C₆-cycloalkyl, or a4-6-membered heterocycloalkyl, wherein said C₄-C₆-cycloalkyl or4-6-membered heterocycloalkyl may be optionally substituted with one tothree substituents independently selected from halo, —OH, oxo, and—C₁-C₃ alkyl, wherein said 5 or 6-membered heteroaryl, or phenyl may beoptionally substituted with one to three substituents independentlyselected from halo, —CN, —OH, —O—C₁-C₃ alkyl and —C₁-C₃ alkyl; and R⁴,R⁵, R⁶, and R⁷ are selected from the group consisting of: H, —OH,C₃-cycloalkyl, —C₁-C₃-alkyl, —(C₁-C₃-alkyl)-S—(C₁-C₃-alkyl),—S—C₁-C₃-alkyl, —(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl,—C(O)O—C₁-C₃-alkyl, —C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, wherein R³⁰and R³¹ are each independently selected from the group consisting of: H,and —C₁-C₃ alkyl, —O—C₁-C₃-alkyl, and halo.

In certain embodiments, R¹ is a thieno[3,2-c]pyridinyl, which may beoptionally substituted as specified herein. The positions of athieno[3,2-c]pyridine are numbered as follows:

A thieno[3,2-c]pyridinyl is a monovalent radical ofthieno[3,2-c]pyridine. Thus, in certain embodiments of the presentinvention, are compounds of formula II:

wherein R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ have any of the values specifiedherein, and wherein the thieno[3,2-c] radical is attached at any ofpositions 2, 3, 4, 6, or 7.

In certain embodiments, R¹ is a thieno[2,3-c]pyridinyl, which may beoptionally substituted as specified. The positions of athieno[2,3-c]pyridine are numbered as follows:

A thieno[2,3-c]pyridinyl is a monovalent radical ofthieno[2,3-c]pyridine. Thus, in certain embodiments of the presentinvention, are compounds of formula III:

wherein R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ have any of the values specifiedherein, and wherein the thieno[2,3-c] radical is attached at any ofpositions 2, 3, 4, 5, or 7.

In certain embodiments, R¹ is a thieno[2,3-b]pyridinyl, which may beoptionally substituted as specified herein. The positions of athieno[2,3-b]pyridine are numbered as follows:

A thieno[2,3-b]pyridinyl is a monovalent radical ofthieno[2,3-c]pyridine. Thus, in certain embodiments of the presentinvention, are compounds of formula IV:

wherein R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ have any of the values specifiedherein, and wherein the thieno[2,3-b] radical is attached at any ofpositions 2, 3, 4, 5, or 6.

In certain embodiments, R¹ is a thieno[3,2-b]pyridinyl, which may beoptionally substituted as specified herein. The positions of athieno[3,2-b]pyridine are numbered as follows:

A thieno[3,2-b]pyridinyl is a monovalent radical ofthieno[3,2-c]pyridine. Thus, in certain embodiments of the presentinvention, are compounds of formula V:

wherein R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ have any of the values specifiedherein, and wherein the thieno[3,2-b] radical is attached at any ofpositions 2, 3, 5, 6, or 7.

In certain embodiments, R¹ is a thieno[3,2-c]pyridinyl or athieno[2,3-c]pyridinyl.

In certain embodiments, R¹ is a thieno[3,2-c]pyridinyl or athieno[2,3-c]pyridinyl, which may be optionally substituted with one tothree substituents each independently selected from the group consistingof: C₁-C₃-alkyl, —(C₁-C₃-alkyl)-S—(C₁-C₃-alkyl), —S—C₁-C₃-alkyl,—(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl, —C(O)O—C₁-C₃-alkyl,—C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, wherein R³⁰ and R³¹ are eachindependently selected from the group consisting of: H, and C₁-C₃alkyl-OH, C₁-C₃-alkyl, halo, and —O—C₁-C₃-alkyl;

In certain embodiments, R¹ is a thieno[3,2-c]pyridinyl or athieno[2,3-c]pyridinyl, which may be optionally substituted with one tothree substituents each independently selected from the group consistingof: —OH, C₁-C₃ alkyl, halo, and —O—C₁-C₃ alkyl.

In certain embodiments, R² and R³ are taken together to form aC₄-C₈-cycloalkyl, or a 4-6-membered heterocycloalkyl, wherein saidC₄-C₆-cycloalkyl or 4-6-membered heterocycloalkyl may be optionallysubstituted with one to three substituents independently selected fromoxo and C₁-C₃ alkyl. In other embodiments, R² and R³ are taken togetherto form a C₅-cycloalkyl, or a 4-6-membered heterocycloalkyl, whereinsaid 4-6-membered heterocycloalkyl is selected from the group consistingof: a tetrahydrofuranyl, a tetrahydrothienyl, a imidazolidinyl, anoxazolidinyl, an imidazolinyl, an isoxazolidinyl, and a pyrrolidinyl. Inother embodiments, R² and R³ are taken together to form a C₅-cycloalkyland R¹ is a thieno[3,2-c]pyridinyl or a thieno[2,3-c]pyridinyl.

In certain embodiments, a compound of the present invention is(2-(6-methylpyridin-2-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thieno[3,2-c]pyridine,or a pharmaceutically acceptable salt thereof.

In certain embodiments, a compound of the present invention is2-(2-(6-methylpyridin-2-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thieno[2,3-c]pyridine,or a pharmaceutically acceptable salt thereof.

In other embodiments, R² and R³ are independently selected from thegroup consisting of: hydrogen, C₁-C₃-alkyl,—(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl, —C(O)O—C₁-C₃-alkyl,—C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, and a C₃-C₆-cycloalkyl, whereinR³⁰ and R³¹ are each independently selected from the group consistingof: H, and C₁-C₃ alkyl. In particular embodiments, R² and R³ areindependently selected from the group consisting of: hydrogen, and C₁-C₃alkyl. In more particular embodiments, R² is C₁-C₂ alkyl and R³ ishydrogen.

In certain embodiments, R⁵, R⁶, and R⁷ are H, and R⁴ is C₁-C₃-alkyl. Inyet other embodiments, R⁴ is methyl. In certain embodiments, R⁵, R⁶, andR⁷ are H, and R⁴ is methyl.

In certain embodiments, R¹ is a thieno[3,2-c]pyridinyl or athieno[2,3-c]pyridinyl, which may be optionally substituted with one tothree substituents each independently selected from the group consistingof: —OH, C₁-C₃ alkyl, halo, and —O—C₁-C₃ alkyl. In certain embodiments,R¹ is a thieno[3,2-c]pyridin-2-yl or a thieno[2,3-c]pyridin-2-yl, whichmay be optionally substituted with one to three substituents eachindependently selected from the group consisting of: —OH, C₁-C₃ alkyl,halo, and —O—C₁-C₃ alkyl. In certain embodiments, R¹ is athieno[3,2-c]pyridine-2-yl, which may be optionally substituted with oneto three substituents each independently selected from the groupconsisting of: —OH, C₁-C₃ alkyl, halo, and —O—C₁-C₃ alkyl. In particularembodiments, R¹ is thieno[3,2-c]pyridinyl-2yl.

In certain embodiments,

In certain embodiments, R⁵, R⁶, and R⁷ are H; R⁴ is a C₁-C₃-alkyl; andR¹ is a thieno[3,2-c]pyridinyl or a thieno[2,3-c]pyridinyl, which may beoptionally substituted with one to three substituents each independentlyselected from the group consisting of: —OH, C₁-C₃ alkyl, halo, and—O—C₁-C₃ alkyl. In certain embodiments, R⁵, R⁶, and R⁷ are H; R⁴ ismethyl; and R¹ is a thieno[3,2-c]pyridinyl or a thieno[2,3-c]pyridinyl,which may be optionally substituted with one to three substituents eachindependently selected from the group consisting of: —OH, C₁-C₃ alkyl,halo, and —O—C₁-C₃ alkyl. In certain embodiments, R⁵, R⁶, and R⁷ are H;R⁴ is methyl; and R¹ is a thieno[3,2-c]pyridinyl or athieno[2,3-c]pyridinyl. In certain embodiments, R⁵, R⁶, and R⁷ are H; R⁴is methyl; and R′ is a thieno[2,3-c]pyridinyl. In certain embodiments,R⁵, R⁸, and R⁷ are H; R⁴ is methyl; and is a thieno[3,2-c]pyridinyl.

In certain embodiments, R⁵, R⁶, and R⁷ are H; R⁴ is methyl; R¹ is athieno[2,3-c]pyridinyl; and R² and R³ are independently selected fromthe group consisting of: hydrogen, and C₁-C₃ alkyl.

In certain embodiments, R⁵, R⁶, and R⁷ are H; R⁴ is methyl; R¹ is athieno[3,2-c]pyridinyl; and R² and R³ are independently selected fromthe group consisting of: hydrogen, and C₁-C₃ alkyl.

In certain embodiments, R⁵, R⁶, and R⁷ are H; R⁴ is methyl; R′ is athieno[2,3-c]pyridin-2-yl; and R² and R³ are independently selected fromthe group consisting of: hydrogen, and C₁-C₃ alkyl.

In certain embodiments, R⁵, R⁶, and R⁷ are H; R⁴ is methyl; R¹ is athieno[3,2-c]pyridin-2-yl; and R² and R³ are independently selected fromthe group consisting of: hydrogen, and C₁-C₃ alkyl.

Examples of compounds of formula I include:

-   2-[1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;-   2-[3,4-dimethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;-   2-[3-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;-   2-[3-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;-   2-[4-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;    and

pharmaceutically acceptable salts thereof.

Another example of a compound of formula I is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;and pharmaceutically acceptable salts thereof. In one particularembodiment, the compound is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine.

Another example of a compound of formula I is2-(4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[2,3-c]pyridine,or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides for methods ofreducing scar formation, comprising administering to a mammal in need ofsuch treatment a therapeutically effective amount of a compound offormula I, or a pharmaceutically acceptable salt thereof. In certainembodiments, the compound of formula I is administered topically. Incertain embodiments, the compound of formula I is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof.

In certain embodiments, the present invention provides for methods ofreducing existing scars, comprising administering to a mammal in need ofsuch treatment a therapeutically effective amount of a compound offormula I, or a pharmaceutically acceptable salt thereof.

In certain embodiments,2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof, may be used in the in themanufacture of a medicament for the inhibition of scar formation.

In another aspect, the present invention provides for methods oftreating a TGFβ-mediated conditions, comprising administering to amammal in need of such treatment a therapeutically effective amount of acompound of formula I, or a pharmaceutically acceptable salt thereof. Incertain embodiments, the TGFβ-mediated condition is selected from thegroup comprising: cancer, breast cancer, lung cancer, colon cancer,prostate cancer, ovarian cancer, pancreatic cancer, melanoma, fibroticdiseases, glomerulonephritis, diabetic nephropathy, hepatic fibrosis,pulmonary fibrosis, arterial hyperplasia, restenosis, scleroderma, anddermal scarring. In certain embodiments, the TGFβ-mediated condition isdermal scarring. In certain embodiments, the compound of formula I is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof. In certain embodiments,the TGFβ-mediated condition is dermal scarring and the compound offormula I is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides for pharmaceuticalcompositions comprising: a therapeutically effective amount of acompound of formula I and a pharmaceutically acceptable excipient. Incertain embodiments, the compound of formula I is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides for topicalpharmaceutical compositions comprising: a therapeutically effectiveamount of a compound of formula I, or pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient suitable fortopical application. In certain embodiments, the compound of formula Iis2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof.

In a further embodiment, the invention is directed to a kit containingat least one of the compounds of the present invention packaged forretail distribution, in association with instructions advising theconsumer on how to use the compound to alleviate a TGFβ-mediatedcondition. An additional embodiment is directed to the use of a compoundas a diagnostic agent to detect inappropriate activation of a TGFβactivated pathway.

DEFINITIONS

As used throughout this application, including the claims, the followingterms have the meanings defined below, unless specifically indicatedotherwise.

The plural and singular should be treated as interchangeable, other thanthe indication of number.

A “scar” is a mark that is present after wound repair at the site of awound. The term “scar” includes keloid scars, hypertrophic scars, andscars that are predominantly not elevated and predominantly do not growbeyond the boundaries of the original wound.

A “keloid scar” is an overgrowth of scar tissue at a wound site, thattypically grows beyond the boundaries of the original wound.

A “hypertrophic scar” is an elevated scar that predominantly does notgrow beyond the boundaries of the original wound.

The term “wound” refers to an injury that disrupts the normal integrityof a tissue, such as skin. A “wound” may intentionally or accidentallyoccur. Examples of wounds include lacerations, contused wounds, closedwounds, open wounds, perforated wounds, incised wounds, puncture wounds,burns, etc.

The terms “compound of Formula I”, “compounds of the invention”, and“compounds” are used interchangeably throughout the application andshould be treated as synonyms.

The term “patient” refers to warm blooded animals such as, for example,guinea pigs, mice, rats, gerbils, cats, rabbits, dogs, monkeys,chimpanzees, stump tail macaques, and humans.

The term “treat” refers to the ability of the compounds to relieve,alleviate, or slow the progression of the patient's disease (orcondition) or any tissue damage associated with the disease.

The term “mammal” refers to a member of the class Mammalia. Examples ofmammals include, without limitation, humans, primates, chimpanzees,rodents, mice, rats, rabbits, horses, livestock, dogs, cats, sheep andcows. In one particular embodiment, a mammal is a human.

The term “isomer” means “stereoisomer” and “geometric isomer” as definedbelow.

The term “stereoisomer” means compounds that possess one or more chiralcenters and each center may exist in the R or S configuration.Stereoisomers include all diastereomeric, enantiomeric and epimericforms as well as racemates and mixtures thereof.

The term “geometric isomer” means compounds that may exist in cis,trans, anti, entgegen (E), and zusammen (Z) forms as well as mixturesthereof.

Certain of the compounds of the formula (I) may exist as geometricisomers. The compounds of the formula (I) may possess one or moreasymmetric centers, thus existing as two, or more, stereoisomeric forms.The present invention includes all the individual stereoisomers andgeometric isomers of the compounds of formula (I) and mixtures thereof.Individual enantiomers can be obtained by chiral separation, by usingavailable synthetic building blocks incorporating the relevantasymmetric center with the appropriate stereochemistry in the synthesis,or by asymmetric synthesis starting with achiral synthetic buildingblocks.

Certain compounds of the present invention may exist as tautomericforms. All such tautomeric forms are included within the scope of thepresent invention.

In addition, the compounds of the present invention can exist inunsolvated as well as solvated forms, including hydrated forms, withpharmaceutically acceptable solvents such as water, ethanol, and thelike. The compounds may also exist in one or more crystalline states,i.e. polymorphs, or they may exist as amorphous solids. All such formsare intended to be encompassed within the scope of the presentinvention.

The term “alkyl group” or “alkyl” means a monovalent radical of astraight or branched chain alkane. For example, a “C₁₋₃ alkyl” is analkyl group having from 1 to 3 carbon atoms. Examples of C₁-C₃straight-chain alkyl groups include methyl, ethyl, and n-propyl.Examples of branched-chain C₁-C₃ alkyl groups include isopropyl. Theterm alkyl includes both “unsubstituted alkyls” and “substitutedalkyls,” the latter of which refers to alkyl groups having substituentsreplacing a hydrogen on one or more carbons (e.g., one to sixsubstituents) of the hydrocarbon backbone. Such substituents may beindependently selected from the group consisting of: halo, I, Br, Cl₂,F, —OH, —COOH, and —NH₂.

Typical substituted C₁-C₃ straight-chain alkyl groups include2-chloropropyl, 2-hydroxy-ethyl, 2-aminopropyl, and trifluoromethyl.

The term “C₃-C₆cycloalkyl” refers to a monovalent radical of amonocyclic alkane containing from 3 to 6 carbons. Examples of“C₃-C₆cycloalkyls” include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

The term “C₄-C₆cycloalkyl” refers to a monovalent radical of amonocyclic alkane containing from 4 to 6 carbons. Examples of“C₄-C₆cycloalkyls” include cyclobutyl, cyclopentyl, and cyclohexyl.

A “4-6-membered heterocycloalkyl” refers to a monovalent radical of a4-6 membered monocyclic heterocycloalkane.

A 4-membered heterocycloalkyl is a 4-membered ring containing 3 carbonsand a heteroatom selected from oxygen, nitrogen, sulfur. The sulfur mayalso be present as S(O) or S(O)₂. Examples of 4-memberedheterocycloalkyl groups include oxetanyl, thietanyl, and azetidinyl.

A 5-membered, heterocycloalkyl contains from 2 to 4 carbon atoms andfrom 1 to 3 heteroatoms selected from the group consisting of: 1 O; 1 S;1 N; 2 N; 1 S and 1 N; 1 S and 2 N; 1 O and 1 N; and 1 O and 2 N,wherein when two O atoms or one O atom and one S atom are present in aring, the two O atoms or one O atom and one S atom are not bondeddirectly to each other. A sulfur may also be present as S(O) or S(O)₂.Examples of 5-membered heterocycloalkyls include tetrahydrofuranyl,tetrahydrothienyl, imidazolidinyl, oxazolidinyl, imidazolinyl,isoxazolidinyl, and pyrrolidinyl.

A “6-membered heterocycloalkyl” contains from 3 to 5 carbon atoms andfrom 1 to 3 heteroatoms selected from the group consisting of: 1 O; 2 O;1 S; 2 S; 1 N; 2 N; 3 N; 1 S, 1 O and 1 N; 1 S and 1 N; 1 S and 2 N; 1 Sand 1 O; 1 S and 2 O; 1 O and 1 N; and 1 O and 2 N, wherein when two Oatoms or one O atom and one S atom are present, the two O atoms or one Oatom and one S atom are not bonded directly to each other. A sulfur mayalso be present as S(O) or S(O)₂. Examples of 6-memberedheterocycloalkyls include tetrahydropyranyl, dioxanyl, 1,3-dioxolanyl,1,4-dithianyl, hexahydropyrimidine, morpholinyl, piperazinyl,piperidinyl, pyrazolidinyl, pyrazolinyl, 1,2,3,6-tetrahydropyridinyl,tetrahydrothiopyranyl, 1,1-dioxo-hexahydro-1λ⁶-thiopyranyl,1,1-dioxo-1λ⁶-thiomorpholinyl, thiomorpholinyl, thioxanyl, and1,3,5-trithianyl.

A “5-membered heteroaryl” is a 5-membered, monocyclic, aromatic ringradial having from 2 to 4 carbon atoms and from 1 to 3 heteroatomsselected from the group consisting of: 1 O; 1 S; 1 N; 2 N; 3 N; 1 S and1 N; 1 S and 2 N; 1 O and 1 N; and 1 O and 2 N. In certain embodiments,a 5-membered heteroaryl is selected from the group consisting offuranyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl,pyrazolyl, pyrrolyl, thienyl, thiazolyl, and triazolyl.

A “6-membered heteroaryl” is a 6-membered, monocyclic, aromatic ringradical having from 4 to 5 carbon atoms and from 1 or 2 heteroatomsselected from the group consisting of: 1 N; and 2 N. In certainembodiments, a 6-membered heteroaryl is selected from the groupconsisting of pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

DETAILED DESCRIPTION OF THE INVENTION Preparation of Compounds

General synthetic schemes for preparing compounds of formula I are setforth below.

Scheme 1 depicts the synthesis of a pyrazole 6. A thieno[3,2-c]pyridine1 (see e.g., Wikel et al. (1993) J. Het. Chem., 30: 289-290) in anaprotic solvent, such as THF (tetrahydrofuran), diethylether, etc. maybe reacted with an alkyl-lithium reagent such as n-butyllithium at orbelow about −40° C. The thieno[3,2-c]pyridine is shown as unsubstitutedin Scheme 1, however it may be optionally substituted as describedherein. Then N-methyl-N-methoxyacetamide 2 (or other suitable acylatingagents such as N-acetyl-morpholine, acetic anhydride, and acetylchloride) is added to the reaction and the reaction is allowed toproceed at −30 to −45° C. to provide the ketone 3 (e.g.,1-(thieno[3,2-c]pyridin-2-yl)ethanone).

The ketone 3 is then reacted with dimethoxy-N,N-dimethylmethanamine(“DMF-DMA”) in DMF (dimethylformamide) at about 70° C. to provide 4(e.g.,(E)-3-(dimethylamino)-1-(thieno[3,2-c]pyridin-2-yl)prop-2-en-1-one). 4is treated with a pyridinyl-hydrazine 5 (e.g.,1-(6-methylpyridin-2-yl)hydrazine) in acetic acid at about 80° C. toyield the regioisomers 6 and 7. The regioisomer 7 can be separated from6 to provide 6 using conventional purification techniques such asprecipitation, filtration, and column chromatography.

Scheme 2 depicts an alternate synthetic route to the pyrazole 6. Asolution of a thieno[3,2-c]pyridine 1 may be reacted under a nitrogengas atmosphere, in a solvent such as THF at about −50° C. to −78° C.with an alkyllithium reagent such as n-butyllithium. Thethieno[3,2-c]pyridine is shown as unsubstituted in Scheme 2, however itmay be optionally substituted as described herein. The addition oftriisopropyl borate and phosphoric acid yields the phosphoric acid saltof the boronic acid 9. The boronate 9 is then coupled to thepyridinyl-pyrazole 10 to provide the pyrazole 6, by the addition of abase such as an inorganic carbonate base (e.g., Na₂CO₃, K₂CO₃, NaHCO₃,etc.) or potassium phosphate tribasic, and a palladium catalyst such asPd(Cl₂)dppf (dichloro (1,1 bis(diphenylphosphino) ferrocene)palladium(11)) and dppf [(1,1 bis(diphenylphosphino) ferrocene. Thereaction may be carried out by refluxing for 1-24 hours in a suitablesolvent such as THF or 1,2-dimethoxyethane; or at about 80-100° C. indioxane. This reaction may also be carried out in the presence of KF andwater. The corresponding boronate esters may be used in place of theboronic acid 9. The group LG of 10 represents a suitable leaving groupsuch as trifluoromethanesulfonyl, Br, I, or Cl. The correspondingthieno[3,2-b]pyridin-2-yl, thieno[2,3-c]pyridin-2-yl, andthieno[2,3-b]pyridin-2-yl analogs of 6 may be prepared usingthieno[3,2-b]pyridine, thieno[2,3-c]pyridine, and thieno[2,3-b]pyridine,respectively, in place of 1.

Pharmaceutically Acceptable Salts

The compounds of the present invention (e.g., compounds of Formula I)may be capable of forming pharmaceutically acceptable salts, includingbut not limited to acid addition and/or base salts. Pharmaceuticallyacceptable salts of the compounds of formula (I) include the acidaddition and base salts (including disalts) thereof. Examples ofsuitable salts can be found for example in Stahl and Wermuth, Handbookof Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH,Weinheim, Germany (2002); and Berge et al., “Pharmaceutical Salts,” J.of Pharmaceutical Science, 1977; 66: 1-19.

Pharmaceutically acceptable acid addition salts of the compounds ofFormula I include non-toxic salts derived from inorganic acids such ashydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic,phosphorus, and the like, as well as the salts derived from organicacids, such as aliphatic mono- and dicarboxylic acids,phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioicacids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Suchsalts thus include the acetate, aspartate, benzoate, besylate(benzenesulfonate), bicarbonate/carbonate, bisulfate, caprylate,camsylate (camphor sulfonate), chlorobenzoate, citrate, edisylate(1,2-ethane disulfonate), dihydrogenphosphate, dinitrobenzoate, esylate(ethane sulfonate), fumarate, gluceptate, gluconate, glucuronate,hibenzate, hydrochloride/chloride, hydrobromide/bromide,hydroiodide/iodide, isobutyrate, monohydrogen phosphate, isethionate,D-lactate, L-lactate, malate, maleate, malonate, mandelate, mesylate(methanesulfonate), metaphosphate, methylbenzoate, methylsulfate,2-napsylate (2-naphthalene sulfonate), nicotinate, nitrate, orotate,oxalate, palmoate, phenylacetate, phosphate, phthalate, propionate,pyrophosphate, pyrosulfate, saccharate, sebacate, stearate, suberate,succinate sulfate, sulfite, D-tartrate, L-tartrate, tosylate (toluenesulfonate), and xinafoate salts, and the like of compounds of Formula I.Also contemplated are the salts of amino acids such as arginate,gluconate, galacturonate, and the like.

Acid addition salts of the basic compounds may be prepared by contactingthe free base form with a sufficient amount of the desired acid toproduce a particular salt. The free base form may be regenerated bycontacting the salt form with a base and isolating the free base. Thefree base forms may differ from their respective salt forms somewhat incertain physical properties such as solubility in polar solvents.

Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metal hydroxides, orof organic amines. Examples of metals used as cations are aluminum,calcium, magnesium, potassium, sodium, and the like. Examples ofsuitable amines include arginine, choline, chloroprocaine,N,N′-dibenzylethylenediamine, diethylamine, diethanolamine, diolamine,ethylenediamine (ethane-1,2-diamine), glycine, lysine, meglumine,N-methylglucamine, olamine, procaine (benzathine) and tromethamine.

The base addition salts of acidic compounds may be prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid. The free acid forms may differ from theirrespective salt forms somewhat in certain physical properties such assolubility in polar solvents.

Pharmaceutical Compositions

Generally, compounds of the present invention may be administered as apharmaceutical composition, comprising one or more pharmaceuticallyacceptable excipients. The phrase “pharmaceutical composition” refers toa composition suitable for administration in medical or veterinary use.The phrase “therapeutically effective amount” means an amount of acompound, or a pharmaceutically acceptable salt thereof, sufficient toinhibit, halt, or allow an improvement in the disease being treated whenadministered alone or in conjunction with another pharmaceutical agentor treatment in a particular subject or subject population. For examplein a human or other mammal, a therapeutically effective amount can bedetermined experimentally in a laboratory or clinical setting, for theparticular disease and subject being treated.

It should be appreciated that determination of proper dosage forms,dosage amounts and routes of administration is within the level ofordinary skill in the pharmaceutical and medical arts and is describedbelow.

The term “excipient” is used herein to describe any ingredient otherthan the compound(s) of the invention. The choice of excipient typicallydepends to a large extent on factors such as the particular mode ofadministration, the effect of the excipient on solubility and stabilityand the nature of the dosage form. Pharmaceutically acceptableexcipients are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington: The Science and Practice of Pharmacy, 20th ed.,Gennaro at al. Eds., Lippincott Williams and Wilkins, 2000).

A compound of the present invention can be formulated as apharmaceutical composition in the form of a syrup, an elixir, asuspension, a powder, a granule, a tablet, a capsule, a lozenge, atroche, an aqueous solution, a cream, an ointment, a lotion, a gel, anemulsion, etc.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable excipients are typicallysolid and liquid excipients. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories and dispersiblegranules. A solid excipient can be one or more substances which may alsoact as diluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material.

In powders, the excipient is typically a finely divided solid which isin a mixture with the finely divided active component. In tablets, theactive component is mixed with the excipient having the necessarybinding properties in suitable proportions and compacted in the shapeand size desired.

The powders and tablets typically contain from 1% to 95% (w/w) of theactive compound. In certain embodiments, the active compound ranges from5% to 70% (w/w). Suitable excipients are magnesium carbonate, magnesiumstearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, a lowmelting wax, cocoa butter and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as an excipient providing a capsule in which theactive component with or without other excipients, is surrounded by aexcipient, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture may then be poured into convenient sized molds,allowed to cool and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. Liquidpreparations can be prepared by dissolving the active ingredient in anaqueous or non-aqueous pharmaceutically acceptable solvent, which mayalso contain suspending agents, sweetening agents, flavoring agents, andpreservative agents as are known in the art.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents and thelike.

A compound of the present invention, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane nitrogen and the like.

A topical composition according to the present invention can be in theform of solutions, lotions, salves, creams, ointments, liposomes,sprays, gels, foams, roller sticks, or any other formulation routinelyused to deliver a topical pharmaceutical composition.

Formulations suitable for parenteral administration, such as, byintravenous, intramuscular, intradermal and subcutaneous routes, includeaqueous and non-aqueous, isotonic sterile injection solutions, which cancontain antioxidants, buffers, bacteriostats and solutes that render theformulation isotonic with the blood of the intended recipient andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers and preservatives.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules and powders in vials orampules. Also, the unit dosage form can be a capsule, tablet, cachet, orlozenge itself, or it can be the appropriate number of any of these inpackaged form. The formulations of compounds can be presented inunit-dose or multi-dose sealed containers, such as ampules and vials.

The compositions containing a compound of the present invention may bepackaged for retail distribution (i.e., an article of manufacture). Sucharticles may be labeled and packaged in a manner to instruct the patienthow to use the product. Such instructions may include the condition tobe treated, duration of treatment, dosing schedule, etc.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 1000 mg, preferably 1.0 mg to 100 mg,or from 0.01% to 95% (w/w) of a unit dose, according to the particularapplication and the potency of the active component. The doseadministered to a subject, in the context of the present inventionshould be sufficient to affect a beneficial therapeutic response in thesubject over time. The composition can, if desired, also contain othercompatible therapeutic agents.

Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. The dose will typically be determined bythe efficacy of the particular compound employed and the condition ofthe subject, the severity of the disease being treated, as well as thebody weight or surface area of the subject to be treated. The size ofthe dose also will be determined by the existence, nature and extent ofany adverse side-effects that accompany the administration of aparticular compound in a particular subject. In determining theeffective amount of the compound to be administered in the treatment orprophylaxis of the disease being treated, the physician can evaluatefactors such as the circulating plasma levels of the compound, compoundtoxicities, and/or the progression of the disease, etc. In addition,compounds of the present invention can be administered at a ratedetermined by factors that can include the pharmacokinetic profile ofthe compound, contraindicated drugs and the side-effects of the compoundat various concentrations, as applied to the mass and overall health ofthe subject.

Generally, treatment is initiated with smaller dosages, which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect undercircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day, if desired.

Methods of Use

While a compound of the present invention may be most typically used toreduce scarring, the invention is not limited to this specificcondition. A compound of the present invention may be used to reduce anexisting scar. A compound of the present invention may also be used toalleviate any type of TGFβ-mediated condition. Examples of theTGFβ-mediated conditions include all types of cancer (e.g., breast,lung, colon, prostate, ovarian, pancreatic, melanoma, all hematologicalmalignancies, etc.), as well as all types of fibrotic diseases (e.g.,glomerulonephritis, diabetic nephropathy, hepatic fibrosis, pulmonaryfibrosis, arterial hyperplasia and restenosis, scleroderma, and dermalscarring). In one particular embodiment, the TGFβ-mediated condition isdermal scarring.

The term “administering” refers to the method of contacting a compoundwith a subject. Thus, the compounds of the present invention can beadministered by a variety of routes including injection, that is,intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, parentally, or intraperitoneally. Also, the compoundsdescribed herein can be administered by inhalation, for example,intranasally. Additionally, the compounds of the present invention canbe administered transdermally, topically, and via implantation. Incertain embodiments, the compounds of the present invention aredelivered orally. The compounds can also be delivered rectally, bucally,intravaginally, or ocularly.

When administered to reduce scarring, a compound of the presentinvention may be typically applied to the wound and/or area around awound. For example, a compound of the present invention can beadministered topically to a wound in the form of a patch, solution,lotion, salve, cream, ointment, liposome, spray, gel, foam, rollerstick, or any other formulation routinely used to deliver a topicalpharmaceutical composition. Alternatively, a compound of the presentinvention may be administered via injection into a wound or area arounda wound. In certain embodiments, a compound of the present invention isadministered to a wound that has been sutured, stapled, taped, and/orbandaged, etc. In certain embodiments, a compound of the presentinvention is administered immediately after the wound occurs. In otherembodiments, a compound of the present invention is administered within1 hour, 8 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1month, 6 months, 1 year, or longer than 1 year, after wound occurrence.In one particular embodiment, the wound occurs via an incision. Incertain embodiments, a therapeutically effective amount of a compound ofthe present invention, or a pharmaceutically acceptable salt thereof maybe administered to a mammal in need of such treatment to inhibit keloidscar formation. In certain embodiments, a therapeutically effectiveamount of a compound of the present invention, or a pharmaceuticallyacceptable salt thereof may be administered to a mammal in need of suchtreatment to inhibit hypertrophic scar formation. In certain embodimentsare methods of inhibiting scar formation, comprising administering to amammal in need of such treatment a therapeutically effective amount of acompound of formula I or a pharmaceutically acceptable salt thereof,wherein the scar would occur on the skin.

Reduction of scarring or inhibition of scar formation is meant to conveythat the compounds of the present invention relate to reducing theappearance of a scar as judged by the patient or a health carepractitioner (e.g., a physician). Reduction of scarring or inhibition ofscar formation may be accompanied by an improvement in one or more ofthe following indicia, including, reduction of extracellular matrixdeposition during healing, reduction of collagen deposition, reductionof size, reduction of shape, reduction of thickness, reduction ofsurface area, reduction of severity, reduction of height, improvement incoloration etc.

In a typical embodiment, the compound is administered topically. As usedherein, topical refers to application of the compounds (and optionalcarrier) directly to the skin or wound area. Topical administration isespecially appropriate for wounds to the skin. The dose may vary, but asa general guideline, the compound may be present in a pharmaceuticallyacceptable carrier in an amount of from about 0.01 to 50 w/w %, and moretypically from about 0.1 to 10 w/w %. The pharmaceutical composition maybe applied to the affected area from 1 to 4 times daily.

In further embodiments of the invention, the compound can beco-administered with other compounds, agents, or dressings to furtherenhance its activity, or to minimize potential side effects. As used inthis application, co-administered refers to administering the compoundof Formula I with a second medicinal, typically having a differingmechanism of action, using a dosing regimen that promotes the desiredresult. This can refer to simultaneous dosing, dosing at different timesduring a single day, or even dosing on different days. The compounds canbe administered separately or can be combined into a single formulation.For the reduction of scarring, agents, compounds, or dressings may beco-administered with a compound of the present invention, includingTGFβ₃, TGFβ₁ antibodies, TGFβ₂ antibodies, triamcinolone acetonide(9-fluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one), steroids,corticosteroids, antibiotics, topical antibiotics, and siliconesheeting. In addition, agents, compounds, or dressings may beco-administered to a wound that improve wound healing, including5-fluorouracil, estrogen, nACh (nicotinic acetylcholine) receptoragonists, FGF (fibroblast growth factor), EGF (epidermal growth factor),IGF (insulin-like growth factor), and PDGF (platelet-derived growthfactor).

In addition, the following therapeutic agents may be co-administeredwith a compound of the present invention to treat a TGFβ-mediatedcondition. Examples of suitable therapeutic agent(s) include, but arenot limited to, standard non-steroidal anti-inflammatory agents(hereinafter NSAID's) (e.g, piroxicam, diclofenac), propionic acids(e.g., naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen),fenamates (e.g., mefenamic acid, indomethacin, sulindac, apazone),pyrazolones (e.g., phenylbutazone), salicylates (e.g., aspirin), COX-2inhibitors (e.g., celecoxib, valdecoxib, and etoricoxib), analgesics andintraarticular therapies (e.g., corticosteroids) and hyaluronic acids(e.g., hyalgan and synvisc), anticancer agents (e.g., endostatin andangiostatin), cytotoxic drugs (e.g., adriamycin, daunomycin,cis-platinum, etoposide, taxol, taxotere), alkaloids (e.g.,vincristine), and antimetabolites (e.g., methotrexate), cardiovascularagents (e.g., calcium channel blockers), lipid lowering agents (e.g.,statins), fibrates, beta-blockers, ACE inhibitors, Angiotensin-2receptor antagonists and platelet aggregation inhibitors, CNS agents(e.g., as antidepressants (such as sertraline)), anti-Parkinsonian drugs(e.g., deprenyl, L-dopa, Requip, Mirapex), MAOB inhibitors (e.g.,selegine and rasagiline), comP inhibitors (e.g., Tasmar), A-2inhibitors, dopamine reuptake inhibitors, NMDA antagonists, Nicotineagonists, Dopamine agonists and inhibitors of neuronal nitric oxidesynthase), anti-Alzheimer's drugs (e.g., donepezil, tacrine, COX-2inhibitors, propentofylline or metryfonate), osteoporosis agents (e.g.,roloxifene, droloxifene, lasofoxifene or fosomax), and immunosuppressantagents (e.g., FK-506 and rapamycin).

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice of theart. The following examples and biological data are being presented inorder to further illustrate the invention. This disclosure should not beconstrued as limiting the invention in any manner.

EXAMPLES

Example 12-(1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[3,2-c]pyridine Stepi: Thieno[3,2-c]pyridine

Thieno[3,2-c]pyridine (9.20 g; dark brown orange solid; see Wikel et al.(1993) J. Het. Chem., 30: 289-290) was dissolved in CH₂Cl₂ and subjectedto column chromatography (Biotage Horizon system, 120 g Isco RediSepcolumn, equilibrate with hexanes, elute with 45% EtOAc (“ethylacetate”)/hexanes, isocratic). The collected fractions were a paleyellow oil that concentrated in vacuo to provide 8.48 g of a solid.

Alternatively, thieno[3,2-c]pyridine was also purified as follows:thieno[3,2-c]pyridine (84.6 g) was dissolved in approximately 180 mL ofdichloromethane. The solution was split into three approximately evenportions. Each portion was applied to a flash silica cartridge (AnalogixSuperFlash SF65-400) and was eluted with an isocratic method (45% or 50%ethyl acetate in hexanes). The desired fractions were concentrated toprovide thieno[3,2-c]pyridine as a light-yellow solid. Three combinedchromatographies afforded a total of 75 g of the title compound; ¹H NMR(400 MHz, CDCl₃) δ 9.12 (d, 1H, J=1.0 Hz), 8.44 (d, 1H, J=5.7 Hz), 7.79(dt, 1H, J=5.7, 1.0 Hz), 7.47 (m, 1H), 7.43 (m, 1H); MS (APCI⁺) m/z 136(MH⁺).

Step ii: 1-(thieno[3,2-c]pyridin-2-yl)ethanone

A solution of thieno[3,2-c]pyridine (5.0 g) in 75 mL THF(tetrahydrofuran) was cooled to −45° C. in a CH₃CN/dry ice bath.n-Butyllithium (1.6M in hexanes) (35.0 ml) was added dropwisemaintaining the internal temperature at or below −40° C. The additiontook about 45 minutes. The reaction was stirred for 1 hour at −45° C.N-methyl-N-methoxyacetamide in 5 mL THF was added and as a result thereaction warmed to −30° C. The reaction was stirred at −45° C. for 1.5hours. The reaction was quenched with saturated NH₄Cl. The aqueous layerhad a pH of about 8. The aqueous layer was extracted three times intoEtOAc. The combined organic extracts were washed with brine and driedover MgSO₄. The material was filtered, concentrated on a rotaryevaporator and subjected to column chromatography (Biotage Horizonsystem, 120 g Analogix column, equilibrate with hexanes, elute with 50%EtOAc/hexanes). The most pure fractions were concentrated on a rotaryevaporator and then triturated with Et₂O to remove some color andcollect a pale orange solid by filtration (2.667 g).

Step iii:(E)-3-(dimethylamino)-1-(thieno[3,2-c]pyridin-2-yl)prop-2-en-1-one

N,N-dimethylformamide-dimethylacetal (DMF-DMA) (9.5 ml) was added to asolution of 1-(thieno[3,2-c]pyridin-2-yl)ethanone (3.17 g) in 20 mL DMF.The reaction was heated to 70° C. overnight. The reaction was thencooled to room temperature. The DMF was removed on a rotary evaporatorto obtain a dark brown orange solid. The residue was diluted with EtOAcand water. A large amount of yellow solid was evident. This wascollected by filtration (3.54 g). This solid was dried in a 50° C.vacuum oven.

Step iv:2-(1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[3,2-c]pyridine

(E)-3-(dimethylamino)-1-(thieno[3,2-c]pyridin-2-yl)prop-2-en-1-one (3.69g) and 1-(6-methylpyridin-2-yl)hydrazine (3.3 g) (which may besynthesized as described below for Example 7, Step ii) was dissolved in40 mL acetic acid. The reaction was heated to 80° C. for 4.5 hours. Thereaction was cooled to room temperature and the solvent was removed on arotary evaporator.

The deep orange red residue was dissolved in EtOAc and saturated NaHCO₃was added until the aqueous layer was pH about 7-8 (about 250 mL). Theaqueous layer was extracted four times into EtOAc. The combined organicextracts were washed with brine and dried over MgSO₄. The material wasfiltered, concentrated, and subjected to column chromatography (BiotageHorizon system, 80 g Isco RediSep column, 0-50%, hold at 50%, 50-75%,hold at 75% EtOAc/hexane).

The fractions containing the desired material were combined andconcentrated. The resulting material was dissolved in hot MeOH and aprecipitate formed on cooling to room temperature. After one hour atroom temperature, the flask was stored at 4° C. for 3 hours. The solidwas collected by filtration, rinsing with hexanes giving 2.79 g. Thematerial was dissolved in boiling acetonitrile and allowed to cool toroom temperature. Once a precipitate became evident the precipitate wascollected by filtration. The precipitate was the undesired regioisomer(2-(1-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)thieno[3,2-c]pyridine).This process was repeated four times to enrich the acetonitrile filtratewith desired regioisomer(2-(1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[3,2-c]pyridine). Theacetonitrile filtrate on cooling contained a white precipitate that wasthe desired product. A total yield of 1.21 g of desired product wasobtained(2-(1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[3,2-c]pyridine).

Example 22-[3-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridineStep i: Thieno[3,2-c]pyridin-2-yl boronic acid

A three neck flask fitted with an internal thermometer containingthieno[3,2-c]pyridine (5.0 g, 37 mmol, 1 equivalent) was evacuated andthen filled with a nitrogen gas atmosphere. THF (60 mL) was added andthe solution was cooled to −44° C. (CH₃CN/dry ice). n-Butyllithium(1.6M/hexane, 25 mL, 41 mmol, 1.1 equivalents) was added over 10minutes, while maintaining the internal temperature at or below −33° C.The reaction was stirred at −33 to −45° C. for 60 minutes. Triisopropylborate (10.2 mL, 44 mmol, 1.2 equivalents) was added and the coolingbath removed. The reaction was allowed to proceed for 105 minutes. Then3.0 mL phosphoric acid (85% aqueous, 3.0 mL, 44 mmol, 1.2 equivalents)was added. A yellow solid formed, which was diluted with water and Et₂O(about 150 mL each). A yellow solid was collected by filtration and wasdried by suction overnight (5.05 g). Analytical Data: ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 7.95 (1H, s), 8.09 (1H, d, J=5.9 Hz), 8.34 (1H, d,J=6.1 Hz), 9.09 (1H, s). MS (APCI, M+1) 180.1. Microanalysis forC₇H₆BNSO₂H₃PO₄: calculated C, 30.35; H, 3.27; N, 5.06; P, 11.18. Found:C, 41.12; H, 3.19; N, 6.53; P, 0.68%.

Step ii. 3-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-ol

Ethyl acetoacetate (10 mL, 78 mmol) and(6-methyl-pyridin-2-yl)-hydrazine (10.1 g, 82 mmol) were combined in 100mL acetic acid. The reaction was heated to 80° C. for 4 hours and 15minutes. The reaction was cooled to room temperature. The solvent wasremoved on a rotary evaporator to provide a dark residue, which wasdissolved in EtOAc. The solution was washed twice with saturated NaHCO₃,then once with water, and then once with brine. A reddish brown solidbecame evident in the biphasic mixture. The biphasic mixture wasfiltered, collecting the solid. The solid was washed with Et₂O toprovide a first solid (6.02 g). The layers of the filtrate wereseparated using a separatory funnel. The organic layer was dried overMgSO₄. The material was filtered and concentrated by rotary evaporation.The resulting brown solid was triturated with Et₂O and subjected tofiltration to provide a second solid (1.98 g) and a first filtrate. Asolid precipitated out of the first filtrate, which was collected bypouring the filtrate into another filter to provide 1.69 g of a thirdsolid. The first filtrate and the 1.98 g (second solid) wereconcentrated to dryness. The resulting solid was dissolve in hot EtOAcand hexanes were added until a cloudiness persisted. The solution wasallowed to stand overnight. A very dark solid was isolated by filtrationand subjected to column chromatography (Biotage Horizon system, 35 gAnalogix column, 30% EtOAc/hexane). A yellow solid was isolated that wasdissolved in hot EtOAc and hexanes were added until a cloudinesspersisted. The solution was allowed to stand overnight. The motherliquor was decanted and the solid was dried solid in 50° C. vacuum ovenfor several hours to provide a fourth solid. The first solid (6.02 g),third solid (1.69 g), and fourth solid (4.03 g) were combined to provide11.74 g of the title compound.

Step iii. 3-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yltrifluoromethanesulfonate

A mixture of 3-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-ol (10.0 gin CH₂Cl₂ (100 mL) and Et₃N (triethylamine) (8.1 mL) was cooled at −70°C.

Triflic anhydride (9.8 mL) was added over 10 minutes while maintainingthe temperature below −50° C. The reaction suspension was warmed to roomtemperature and became a solution. The solution was concentrated toremove most of the CH₂Cl₂. The solution was loaded directly onto anAnalogix SuperFlash SF65-400; using a Biotage auto fraction collector;EtOAc/hexanes (20/80). Fractions containing a colorless liquid wereisolated (9.46 g).

Step iv:2-[3-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine

In a 100 mL three neck round bottom flask fitted with a reflux condenserwas added 3-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yltrifluoromethanesulfonate (1.16 g), thieno[3,2-c]pyridin-2-ylboronicacid (1.0 g), potassium phosphate tribasic (2.3 g) and 30 mL dioxane.The flask was evacuated under a vacuum and the suspension bubbled withnitrogen gas. To this was added PdCl₂(dppf) (Strem,dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct, 0.60 g). Again the flask was evacuated under avacuum and the suspension bubbled with nitrogen gas. The reaction washeated to 100° C. and permitted to run overnight. The reaction wascooled to room temperature and filtered through a pad of Celite, elutingwith EtOAc (˜600 mL). The EtOAc filtrate was concentrated to dryness onrotary evaporator. The residue was diluted with CH₂Cl₂ and a beige solidwas filtered off. The CH₂Cl₂ filtrate was purified by columnchromatography (Biotage Horizon system, 34 g Analogix column, 0-100%EtOAc/hep, hold at 100% EtOAc followed by 10% MeOH/EtOAc). The fractionscontaining desired product were combined and concentrated on the rotaryevaporator to give a dark oil.

The dark oil (about 800 mg) was subjected to preparative columnchromatography on a Phenomenex, Gemini C-18 column (150×19 mm, 5 □M;Mobile Phase A: Water (+0.1% NH₄OH), B: CH₃CN (+0.1% NH₄OH); Gradient:90-10% A over 10 min, hold at 90% A for 1.5 minutes; Flow Rate: 28mL/min; Injection Volume: 2 mL; Detection: DAD 210-350 nm, MS APCI⁺, MSAPCI⁻).

The desired fractions were dried down in a rotary evaporator. The solidwas dissolved in hot EtOH and then 2-5 mLs of heptane was added. Afterstanding, the material was filtered.

The filtrate was concentrated to dryness. Diethylether was added and thesolution was filtered on a sintered glass funnel. Additionaldiethylether was used to wash the material until <10 mg of brown solidremained on the sintered glass funnel. The filtrate was concentrated todryness giving an off-white solid.

The off-white solid was dissolved in hot EtOH (about 1-2 mL) and dilutedwith heptane until a cloudiness persisted, and was permitted to stand.Needles (slight yellow color) then started to precipitate from thesolution. After about 3 hours, the mother liquors were decanted intoanother flask. The needles were dried in a 40° C. vacuum oven for over48 hours to provide 65 mg of a white solid. mp 131-132° C.

Example 32-[3-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridineStep i: 5-ethyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-ol

A stirring mixture of 6-methyl-pyridin-2-yl)-hydrazine (10.0 g) andethyl propionylacetate (12 mL) in glacial acetic acid (100 mL) waswarmed to 80° C. for five hours. The reaction mixture was concentratedunder reduced pressure and the evaporate was treated with saturatedaqueous sodium bicarbonate (200 mL). The mixture was extracted withethyl acetate (200 mL). The organic phase was washed with a freshportion of saturated aqueous sodium bicarbonate (200 mL), water (200mL), and brine solution (200 mL). The organic phase was subsequentlydried over anhydrous sodium sulfate and was concentrated under reducedpressure. The evaporate was purified by flash silica gel columnchromatography. Elution through a silica cartridge (Analogix, 400 g)with a gradient (100% hexanes to 60% ethyl acetate in hexanes) affordedan oil. The oil was reconstituted in chloroform and the solution wasconcentrated under reduced pressure to afford the title compound as anoil (7.2 g); ¹H NMR (400 MHz, CDCl₃) δ 13.07 (broad s, 1H), 7.70 (m,1H), 7.64 (d, 1H, J=8.2 Hz), 6.92 (d, 1H, J=7.4 Hz), 5.42 (s, 1H), 2.59(quartet, 2H, J=7.6 Hz), 2.51 (s, 3H), 1.24 (t, 3H, J=7.6 Hz); ¹³C NMR(100 MHz, CDCl₃) δ 13.5, 22.7, 23.8, 86.8, 108.8, 119.2, 140.2, 154.3,155.0, 157.2, 157.4; MS (APCI⁺) 204 (MH⁺).

Step ii: trifluoro-methanesulfonic acid5-ethyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl ester

To a stirring mixture of5-ethyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-ol (7.1 g) andtriethylamine (5.4 mL) in dichloromethane (75 mL) at −78° C. under anitrogen atmosphere was added trifluoromethanesulfonic anhydride (6.5mL) over 5 minutes. The cold bath was removed and the reaction mixturewas allowed to warm to room temperature and was stirred overnight. Thereaction mixture was concentrated under reduced pressure and theconcentrate was purified by flash silica gel column chromatography.Elution through a silica cartridge (Analogix SuperFlash SF65-400) with agradient (100% hexanes to 35% ethyl acetate in hexanes over 2400 mL)afforded the title compound as an oil (10.38 g) after chloroform chase;¹H NMR (400 MHz, CDCl₃) δ 7.69 (m, 1H), 7.59 (dd, 1H, J=8.2, 0.6 Hz),7.07 (dd, 1H, J=7.6, 0.4 Hz), 6.10 (s, 1H), 2.68 (quartet, 2H, J=7.6Hz), 2.57 (s, 3H), 1.28 (t, 3H, J=7.6 Hz); ¹⁹F NMR (376 MHz, CDCl₃) δ−73.9 (s); MS (APCI⁺) 336 (MH⁺), 204 (MH⁺-SO₂CF₃).

Step iii:2-[3-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine

In a 100 mL three neck round bottom flask fitted with a reflux condenserwas added 3-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yltrifluoromethanesulfonate (1.21 g), thieno[3,2-c]pyridin-2-ylboronicacid (1.0 g), potassium phosphate tribasic (2.3 g) and 30 mL dioxane.The flask was evacuated under a vacuum and the suspension bubbled withnitrogen gas. To this was added PdCl₂(dppf) (Strem,dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct, 0.60 g). Again the flask was evacuated under avacuum and the suspension bubbled with nitrogen gas. The reaction washeated to 100° C. and allowed to proceed overnight. The reaction wascooled to room temperature. The reaction was then filtered throughCelite, rinsing well with EtOAc. The filtrate was concentrated on arotary evaporator and the dark oily material was diluted with CH₂Cl₂. Anoff-white solid was filtered from the CH₂Cl₂. The filtrate was loaded ona column (Biotage Horizon system, 34 g Analogix column, 0-100%hepane/EtOAc), then holding at 100% EtOAc). The desired fractions werecollected to yield a dark oil (about 900 mg). The fractions werechromatographed on a Phenomenex, Gemini C-18 column as described abovein Example 2.

The desired fractions were combined and dried down in a rotaryevaporator to provide a solid sample. The solid sample was dissolved inwarm EtOH. The solution was concentrated to dryness on a rotaryevaporator to obtain a dark residue. The residue was dried in 45° C.vacuum oven for 2 hours. The resulting material was part dark oil andpart solid. Diethylether was added and the material was filtered toprovide a dark brown solid, and a light orange-yellow colored filtrate.The filtrate was concentrated to dryness to obtain a tan solid (231 mg).

Diethylether was added to the tan solid. The material was filteredthrough fluted filter paper, leaving a brown residue on filter paper.The filtrate was concentrated to dryness on the rotary evaporator. Theresulting solids were again dissolved in hot EtOH (1-2 mL) and dilutedwith heptane until a cloudiness persisted. After 2 hours of standing, noprecipitate was evident. The liquid was then concentrated to dryness.While on a rotary evaporator, when the liquid had reduced toapproximately half of the original volume, a brown solid was evident.The liquid was removed from the rotary evaporator and the solution beganto display a white cloudiness. The liquid was filtered into anotherflask using filter paper to remove brown solid. A white solid started toprecipitate in the filtrate. After about 15 minutes, no moreprecipitation was observed, and the material was filtered to obtain asolid. The solid was dried in a 40° C. vacuum oven for about 48 hours toprovide 71 mg. mp 89-90° C.

Example 42-[4-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridineStep i: 4-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-ol

A suspension of KOMe (14.0 g) and ethyl butyrate (19.8 mL) in DMF (15mL) was stirred at room temperature. Ethyl formate (8.1 mL) was addedover 15 minutes, which resulted in much foaming. The reaction wasstirred for an additional hour, and the reaction became a very stirrablemixture with no foamy layer. A solution of1-(6-methylpyridin-2-yl)hydrazine (12.3 g), MeOH (35 mL), and HOAc (5.7mL) were added over 20 minutes. The reaction was exothermic and reacheda temperature of 30° C. after an initial addition. An ice-water bath wasused to keep the reaction below 20° C. for the remaining addition of thesolution. The reaction was heated at reflux for 3 hours and then stirredat room temperature for about 48 hours.

The reaction was cooled to 10° C. with an ice-water bath. The pH wasadjusted to about 7 with HOAc (10 mL) to obtain a thick, unstirrablemass. The reaction was diluted with H₂O (100 mL) to result in a dark redsolution. The solution was extracted with EtOAc (200 mL). The organicphase was washed with H₂O (2×100 mL), dried (MgSO₄) and SiO₂ (30 mL),and filtered through SiO₂ (30 mL). The material was concentrated toprovide 10.50 g of a dark brown oil. The oil was chromatographed usingan Analogix SuperFlash SF65-400 and Biotage auto fraction collector;EtOAc/hexanes (40/60). Fractions containing the desired product werecombined to afford 6.40 g of a yellow-orange liquid.

Step ii: 4-ethyl-11-(6-methylpyridin-2-yl)-1H-pyrazol-5-yltrifluoromethanesulfonate

A solution of 4-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-ol (6.40 g)in CH₂Cl₂ (65 mL) and Et₃N (4.8 mL) was cooled at −70° C. Triflicanhydride (Tf₂O) (5.83 mL) was added over 10 minutes while maintainingthe temperature below −50° C. The reaction is exothermic. The reactionwas stirred for an additional 30 minutes at less than −50° C. and thenwarmed to room temperature. The reaction was then cooled at to less than−50° C. and an additional portion of Tf₂O (1.1 mL; 20%) and Et₃N (1.0mL; 20%) was added. After an hour incubation, then reaction wasconcentrated to remove most of the CH₂Cl₂. The reaction was loadeddirectly onto an Analogix SuperFlash SF40-150; using a Horizon autofraction collector; EtOAc/heptane (20/80). The column was washed withEtOAc/heptane (30/70). Fractions containing the desired product werecollected to provide 6.98 g as a pale yellow liquid.

Step iii:2-[4-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine

In a 100 mL three neck round bottom flask fitted with a reflux condenserwas added 4-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yltrifluoromethanesulfonate (1.2 g), thieno[3,2-c]pyridin-2-ylboronic acid(1.2 g), potassium phosphate tribasic (2.3 g) and 30 mL dioxane. Theflask was evacuated under a vacuum and the suspension bubbled withnitrogen gas. To this was added PdCl₂(dppf) (Strem,Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct, 0.60 g). Again the flask was evacuated under avacuum and the suspension bubbled with nitrogen gas. The reaction washeated to 100° C. overnight. Then 0.295 g PdCl₂(dppf) (Strem,Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct) was added and the reaction was continued for 4hours and 15 minutes. The reaction was cooled to room temperature.

The reaction contents were filtered through Celite rinsing well withEtOAc until the desired product had eluted. The filtrate wasconcentrated to dryness on a rotary evaporator to form a dark residue.The dark residue was taken up in CH₂Cl₂ and the solids were filteredaway. The resulting filtrate was reduced by concentration on a rotaryevaporator before loading the material on a column (Biotage Horizonsystem, 0-100% EtOAc over 700 mL then hold at 100% EtOAc, 500 mL). Thetubes with most product evident had a white solid on the sides of theflask.

The tubes containing desired product were concentrated by rotaryevaporation to afford a dark oil. The dark oil was triturated with Et₂Oand the solid was filtered away. Additional solid precipitate wasisolated from the filtrate and combined with the solid from the originalfiltration. The combined solids were dissolved in hot EtOH (about 2 mL).Heptane was added until a precipitate started to form. A dark solid thenformed. After about 10 minutes, it appeared that the solid had finishedprecipitating. The mixture was filtered through paper filter intoanother flask. The filtrate was diluted with more heptane until acloudiness persisted. The filtrate was allowed to stand overnight atroom temperature. White needles formed in the flask with a dark residueon the bottom of flask. The contents of the flask were concentrated todryness on a rotary evaporator and dissolved in 1-2 mL hot EtOH,filtered through filter paper and diluted with heptane until acloudiness persisted. The flask was allowed to stand for 2 hours. Thenthe mixture was put on a rotary evaporator to reduce the volume of thematerial. While on the rotary evaporator, when approximately half of theoriginal volume was present, a brown solid became evident. The flask wasremoved from the rotary evaporator and filtered into another flask usingfilter paper to remove a brown solid. A white solid started toprecipitate in the filtrate. The white solid was collected by filtrationand dried in a 40° C. vacuum oven for over 48 hours to obtain 85 mg ofthe title compound.

Example 52-(2-(6-methylpyridin-2-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thieno[3,2-c]pyridineStep i: thieno[3,2-c]pyridine-2-boronic acid

To a stirring mixture consisting of thieno[3,2-c]pyridine (2.0 g) inanhydrous tetrahydrofuran (25 mL) at −40° C. was added a solutionconsisting of 1.6 M n-butyllithium in hexanes (10 mL) over severalminutes (dropwise at first followed by a slow, steady stream). Thereaction mixture was stirred for five minutes and triisopropyl borate(4.2 mL) was subsequently added. The cold bath was removed and themixture was allowed to stir for one hour. Water (2 mL) was added to thestirring mixture, which caused a pale yellow solid to precipitateinstantly. The precipitate was collected by vacuum filtration and thesolids were rinsed lightly with water (giving a cloudy yellow filtrate)and subsequently with diethyl ether (giving a dark orange filtrate). Theaqueous portion of the filtrate was separated from the organic solvent,and then diluted with 1,4-dioxane (100 mL) and the solution wasconcentrated under reduced pressure. Addition of 1,4-dioxane (100 mL) tothe concentrate and subsequent concentration under reduced pressure wasrepeated twice to afford a yellow powder (0.8 g) upon final evaporation.

Step ii: 2-[(6-methyl-pyridin-2-yl)-hydrazono]-cyclopentanecarboxylicacid ethyl ester

A solution of ethyl 2-oxocyclopentanecarboxylate (6.34 g, 5.88 mL) and(6-methyl-pyridin-2-yl)-hydrazine (5 g) in ethanol (100 mL) was heatedto 80° C. under an atmosphere of nitrogen for about 19 hours. Thereaction mixture was cooled to room temperature and the solvent removedin vacuo to provide a crude material (7.72 g), which was directly takento the next reaction.

Step iii:2-(6-methyl-pyridin-2-yl)-2,4,5,6-tetrahydro-cyclopentapyrazol-3-ol

A solution of crude2-[(6-methyl-pyridin-2-yl)-hydrazono]-cyclopentanecarboxylic acid ethylester (6.6 g) and sodium methoxide (2.73 g) in methanol (250 mL) wasconcentrated under reduced pressure, nearly to dryness, to provide abrown paste. This paste was then heated to 160° C. for 2 hours beingcautious of excessive bubbling. After 2 hours the reaction was cooled toroom temperature and water (100 mL) was added. The pH was adjusted to 7with 1N HCl, ethyl acetate (200 mL) was then added and the resultantmixture was stirred until precipitate dissolution occurred. The layerswere separated and the aqueous layer was washed with another portion ofethyl acetate (100 mL). The combined organic layers were washed withwater (200 mL) and brine (200 mL) and dried over MgSO₄, filtered, andconcentrated in vacuo to provide of product (3.6 g) to use without anyfurther purification.

Alternatively: The same reaction can be carried out at a lowertemperature of 125° C. by starting with 21.0 g of2-[(6-methyl-pyridin-2-yl)-hydrazono]-cyclopentanecarboxylic acid ethylester to provide 17.3 g of crude product.

Step iv: trifluoro-methanesulfonic acid2-(6-methyl-pyridin-2-yl)-2,4,5,6-tetrahydro-cyclopentapyrazol-3-ylester

To an oven-dried rounded bottomed flask was added2-(6-methyl-pyridin-2-yl)-2,4,5,6-tetrahydro-cyclopentapyrazol-3-ol(17.3 g) and triethylamine (9.76 g, 13.44 mL) in dichloromethane (350mL) and the reaction mixture was cooled to −78° C. To this was addedtrifluoromethane sulfonic anhydride (16.23 mL) via a pressure equalizingfunnel over 10 minutes. The resultant mixture was stirred at −78° C. for1 hour and then allowed to warm to room temperature and stir for anadditional hour. The solvent was removed in vacuo to provide a brownprecipitate. The brown precipitate was purified via flash silica gelchromatography (60% hexanes/40% ethyl acetate) to provide an off-whitesolid (8.848 g) followed by another batch of material (12.2 g) ofslightly less pure material.

Step iv:2-(2-(6-methylpyridin-2-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thieno[3,2-c]pyridine

An oven-dried three-neck round-bottom flask equipped with refluxcondenser and gas inlet valve was charged withthieno[3,2-c]pyridine-2-boronic acid (0.7 g). The flask was evacuatedand purged with nitrogen gas three times. The content of the flask waskept under a nitrogen atmosphere for four days. To the reaction flaskwas added trifluoro-methanesulfonic acid2-(6-methyl-pyridin-2-yl)-2,4,5,6-tetrahydro-cyclopentapyrazol-3-ylester(0.7 g), potassium phosphate tribasic (2.5 g), and 1,4-dioxane (30 mL).The flask was evacuated and purged with nitrogen three times more.Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (0.65 g) and 1,1′-bis(diphenylphosphino)ferrocene(0.43 g) were added and the mixture was degassed and purged withnitrogen three more times. The stirring mixture was brought to refluxovernight. The reaction mixture was diluted with ethyl acetate (250 mL)and the solution was treated with activated carbon and filtered throughCelite.

The resulting filtrate was concentrated under reduced pressure and theconcentrate was purified on a 120 g RediSep flash silica cartridge onthe Biotage system with 100% hexanes to 100% ethyl acetate over 1800 mLfollowed by 100% ethyl acetate until the desired material eluted. Thedesired material was a dark residue. A dark liquid separated from asolid that adhered to the insides of the flask. The liquid was removedwith absolute ethanol by drawing off with a pipet and the dark solutionwas evaporated overnight to a dark viscous residue. The material wasdissolved in acetonitrile (15 mL) and was purified by prep HPLC (Watersprep HPLC system; Stationary phase: Waters DeltPak C18 5 □m, 100Angstom, 300×50 mm I.D., P/N 011801, WAT 011801, No. 330009125W; Mobilephase 80:20 to 40:60 water-acetonitrile with 0.1% formic acid over 30minutes). The combined fractions were combined and concentrated underreduced pressure to give a clear colorless aqueous solution. Thesolution was treated with saturated aqueous potassium carbonate to givea milky suspension. Extraction with one portion of diethyl ether and twoportions of ethyl acetate, drying of the combined extracts overanhydrous potassium carbonate, and concentration under reduced pressuregave a film that began to solidify with time. The film was dissolved inabsolute ethanol and was concentrated under reduced pressure to afford alight-yellow oil that solidified with time. The material was subjectedto high vacuum at room temperature to afford the title compound as alight-yellow solid (21 mg); melting point: solid form morphs from121-126° C., melts at 146-148° C.

Example 62-[3,4-dimethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridineStep I. 3,4-dimethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-ol

To a 250 mL round-bottomed flask were added ethyl 2-methyl acetoacetate(5.0 g, 35 mmol), 1-(6-methylpyridin-2-yl)hydrazine (4.48 g, 36 mmol),and 12 mL acetic acid. The mixture was heated at 80° C. for 9 hours.Water and EtOAc were added to the mixture. The layers were separated.The aqueous layer was extracted with EtOAc. The combined organic layerswere washed with water and brine, dried (MgSO₄), filtered andconcentrated in vacuo (6.68 g).

Step ii. 3,4-dimethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-6-yltrifluoromethanesulfonate

To the 500 mL round-bottomed flask with3,4-dimethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-ol (6.68 g, 33 mmol)were added Et₃N (5.5 mL, 39 mmol) and 35 mL CH₂Cl₂. the solution wascooled in dry ice acetone bath. Tf₂O (6.1 mL, 36 mmol) was added slowly.The mixture was stirred at −78° C. and allowed to warm to roomtemperature. The mixture was then concentrated in vacuo. The crudeproduct was purified using a Biotage Horizon System (0 to 25%EtOAc/hexane) to afford a colorless oil (8.28 g).

Step iii:2-[3,4-dimethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-6-yl]thieno[3,2-c]pyridine

In a 100 mL three neck round bottom flask fitted with a reflux condenserwas added 3,4-dimethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yltrifluoromethanesulfonate (0.90 g. 2.7 mmol, 1.2 equivalents),thieno[3,2-c]pyridin-2-ylboronic acid trihydrogen phosphate (0.72 g, 2.6mmol, 1 equivalent), potassium phosphate tribasic and 20 mL dioxane. Theflask was evacuated under a vacuum and the suspension bubbled withnitrogen gas. To this was added PdCl₂(dppf) (Strem,Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct, 0.54 g, 0.65 mmol, 0.3 equivalents). Again theflask was evacuated under a vacuum and the suspension bubbled withnitrogen gas. The reaction was heated to 100° C. for 16 hours and thencooled to room temperature.

The reaction was filtered through Celite, rinsing well with EtOAc untilall the desired material eluted (about 400 mL). The EtOAc filtrate wasconcentrated to a dark residue. The dark residue was taken up in CH₂Cl₂and the solids were filtered off. The CH₂Cl₂ was subjected to columnchromatography (Biotage Horizon system, 0-100% hepane/EtOAc over 800 mLthen hold at 100% EtOAc, 500 mL). The fractions were concentrated into 3portions: the portion that contained a lightly colored oil (420 mg)partially solidified after standing overnight. The material wasdissolved in hot EtOH and heptane was added. Upon standing overnight nosolid appeared to precipitate. The material was concentrated to dryness.About 15 mL of Et₂O was added and the material was filtered through apaper filter, leaving a minor amount of a tan residue. The residue wasconcentrated to dryness and about 1 mL Et₂O was added. Then about 15 mLheptane was added until the mixture remained cloudy. A precipitate wasnot apparent after standing at 4° C. for about 2 hours. The material wasthen concentrated to dryness to obtain a brown oil. Hepatane was addedand small circles of solid formed, some were off-white, others werebrown. The heptane was decanted and the solid was dried in a 50° C.vacuum oven for about 48 hours to provide 229 mg of a tan solid. mp108-109° C.

Example 72-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridineStep i: Thieno[3,2-c]pyridine 2-boronic acid trihydrogen phosphate

To a stirring mixture of thieno[3,2-c]pyridine (2.0 g) in anhydroustetrahydrofuran (25 mL) at −40° C. was added a solution of 1.6 Mn-butyllithium in hexanes (10 mL) dropwise over five minutes. Thereaction mixture was stirred for ten minutes and triisopropyl borate(4.2 mL) was subsequently added. The cold bath was removed and themixture was allowed to stir for three hours. Phosphoric acid (85%aqueous solution, 1.2 mL) was added to the stirring mixture. Water (10mL) was subsequently added, which caused a pale yellow solid toprecipitate instantly. More water (100 mL) was added and diethyl ether(100 mL) was added. The suspension was vacuum filtered. The solids weresuction dried to afford the title compound as a pale yellow powder (2.73g); microanalysis for C₇H₆BNO₂S.H₃PO₄% C (calc/found) 30.35/31.41, % H,3.27/3.24, % N, 5.06/5.04, % S 11.58/11.93, % P 11.18/8.58; ¹H-NMR (400MHz, DMSO-d₆, CDCl₃, CD₃OD, D₂O mixture) δ 9.01 (d, 1H, J=1.0 Hz), 8.26(d, 1H, J=6.0 Hz), 7.94 (s, 1H), 7.91-7.83 (m, 1H); MS (APCI⁺) m/z 180(MH⁺).

Step ii: 1-(6-methylpyridin-2-yl)hydrazine

A mixture of 2-bromo-6-methyl-pyridine (602.10 g) and hydrazine hydrate(1570 ml) was refluxed at 120° C. for 6 hours, and then stirred at roomtemperature for about 48 hours. The solid separated was extracted withdiethylether (3×1.5 L) and the combined ether extracts were washed withbrine, dried over Na₂SO₄, filtered and concentrated (˜430 g). Theresidue was stirred in a mixture of ether (200 ml) and hexane (1.5 L),filtered and washed with 5% diethylether in hexane to give a pale yellowsolid (sticky). The solid was vacuum distilled at 106-113° C./2 mm,which solidified instantly. The solid was then dissolved in 2 L ofdiethylether and precipitated by adding 2 L of hexane, filtered andvacuum dried to give 1-(6-methylpyridin-2-yl)hydrazine as white solid(240 g).

Alternatively, 1-(6-methyl-pyridin-2-yl)-hydrazine was also synthesizedas follows: A jacketed reaction flask was equipped with mechanicalstirrer, nitrogen inlet, and reflux condenser and the jacket was cooledto 10° C. The reactor was programmed to automatically cool to 0° C. ifat any point the internal temperature exceeded 80° C. The reactor waspurged with a steady stream of nitrogen gas. To the reactor was charged25 g (0.225 mol) of 2-fluoro-6-methylpyridine followed by 100 ml ofisopropanol and the jacket temperature was adjusted to 73° C.(corresponding to an internal temperature of 70° C.). To the reactor wascharged 44 g (0.90 mol) of 65% aqueous hydrazine and the mixture wasstirred overnight with the jacket set at 70° C. The reactor contentswere diluted with 125 ml of methyl t-butyl ether (MTBE) and 13 ml ofsaturated brine to give two layers which were separated. The aqueouslayer was extracted with 2×60 ml of MTBE. The combined organic layerswere combined and dried over sodium sulfate (anhydrous). The dryingagent was removed by filtration and the organic filtrate wasconcentrated by rotary evaporation. The resulting oil solidified uponstanding to give the desired product (21.1 g) as a white solid.

Step iii: 4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-ol

To a stirring mixture of sodium methoxide (13.6 g) in anhydroustetrahydrofuran (50 mL) under a nitrogen atmosphere at room temperaturein an oven-dried 500 mL three-neck reaction vessel was added anhydrousmethyl formate (12.6 mL). The mixture frothed and to the suspension wasadded ethyl formate (28.7 mL) with a slow, steady stream via syringewhile swirling the reaction vessel by hand to facilitate more efficientmixture of the suspension. The subsequent mixture was stirred at roomtemperature for five hours. Glacial acetic acid (14 mL) was added whilethe reaction vessel was swirled vigorously by hand for several minutesto facilitate more efficient mixture. The subsequent suspension wasstirred overnight and was filtered through Celite. The Celite filterplug was washed twice with diethyl ether (2×200 mL) and suctioned. Thefiltrate produced a sparse white solid precipitate that was removed by afurther vacuum filtration through a medium fritted Buchner funnel. Thesubsequent filtrate was concentrated under reduced pressure (water bathtemperature=45° C.) to afford a clear, lightly colored oil(approximately 15 g). The oil possessed the distinct odor of aceticacid. The oil was dissolved in absolute ethanol (50 mL). To thissolution was added solid (6-methyl-pyridin-2-yl)-hydrazine (5.4 g). Thestirring mixture was brought to gentle reflux under a nitrogenatmosphere for four days. The mixture was concentrated under reducedpressure and the afforded orange oil was dissolved in dichloromethane(50 mL). The dichloromethane solution was applied to a 400-g Analogixflash silica cartridge on a Biotage instrument. Elution on the Biotageinstrument with a gradient (100% heptane to 70% ethyl acetate in heptaneover four column volumes, or 2400 mL). The fractions were dried down toafford a solid (7.1 g).

Alternatively, 4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-ol wasalso produced as follows:

To a solution of ethyl formate (117.5 g) and ethyl propionate (54.0 g)in 40 ml of tetrahydrofuran (THF) was added, drop-wise, potassiumt-butoxide (502 ml of a 1.0 molar solution in THF) at ambienttemperature over approximately 0.5 hour. A white precipitate began toform along with some bubbling. After another 7.5 hours the reaction wasfiltered, the solid was washed with diethyl ether and then dried undervacuum at 40° C. for 16 hours to give potassium2-ethoxycarbonyl-propen-1-olate as a grey powder (23.5 g).

To a solution of this potassium salt (17.6 g) in 450 ml of 1-propanolwas added (6-Methyl-pyridin-2-yl)-hydrazine (11.7 g) followed by aceticacid (6.5 ml). After stirring for 10 minutes the mixture was heated atgentle reflux for 5 hours. More acetic acid (5.5 ml) was added andreflux was continued for a further 16 hours.

The solvent was removed under vacuum. The resulting residue was dilutedwith ethyl acetate (350 ml) and the solution was carefully washed withsaturated sodium bicarbonate solution (200 ml) until the remainingacetic acid was neutralized. The phases were separated and the aqueousphase was extracted with ethyl acetate (50 ml). The organic phases werecombined and then washed with water (100 ml), then brine (100 ml) anddried over magnesium sulfate. The drying agent was removed by filtrationand the solvent was removed under vacuum to give the product as anorange solid (16.26 g).

Step iv: Trifluoro-methanesulfonic acid4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl ester

To a stirring mixture of a dichloromethane solution of4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-ol (13.39 g in 50 mL ofdichloromethane) and triethylamine (11 mL) in additional dichloromethane(50 mL more, 100 mL total dichloromethane) at −78° C. under a nitrogenatmosphere was added triflic anhydride (13.3 mL) with a slow, steadystream via syringe over five minutes. The cold bath was removed and themixture was allowed to warm gradually over one hour, thirty minutes toroom temperature. The mixture was concentrated under reduced pressureand the concentrate was purified by flash silica chromatography. Elutionwith a gradient (100% heptane to 30% ethyl acetate in heptane over threecolumn volumes, or 1800 mL, then 30% to 45% ethyl acetate over twoadditional column volumes, or 1200 mL) through a 400-g Analogix flashsilica cartridge on the Biotage instrument afforded the title compoundas a clear colorless oil (18.04 g). ¹H-NMR (400 MHz, CDCl₃) 0.8-7.68 (m,1H), 7.59 (d, J=8.0 Hz, 1H), 7.50 (s, 1H), 7.07 (d, J=7.4 Hz, 1H), 2.56(s, 3H), 2.07 (s, 3H); ¹⁹F-NMR (376 MHz, CDCl₃)

−74.12 (s, 3F); ¹³C-NMR (100 MHz, CDCl₃)

157.9, 151.1, 141.3, 139.5, 139.0, 122.0, 118.6 (quartet, J_(C-F)=321.0Hz), 111.9, 108.7, 23.6, 7.3; MS (APCI⁺) m/z 322.

Step v:2-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine

An oven-dried three-neck round-bottom flask equipped with refluxcondenser and gas inlet valve was charged withthieno[3,2-c]pyridine-2-boronic acid trihydrogen phosphate (1.0 g),trifluoro-methanesulfonic acid4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl ester (1.16 g),potassium phosphate tribasic (2.3 g), 1,4-dioxane (30 mL),dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (0.60 g), and1,1′-bis(diphenylphosphino)ferrocene (0.40 g) and the stirring mixturewas brought to reflux under a nitrogen atmosphere for 18 hours. Thereaction mixture was diluted with ethyl acetate (250 mL) and thesolution was treated with activated carbon and filtered through Celite.The filtrate was concentrated under reduced pressure and the concentratewas purified by silica gel chromatography. Elution through a 120 gRediSep flash silica cartridge on a Biotage system with 100% heptane to100% ethyl acetate over 1800 mL followed by 100% ethyl acetate untilmaterial with the desired mass elutes afforded an oil (1.2 g); MS(APCI⁺) m/z 307 (MH⁺). The product was further purified by dissolving inacetonitrile (30 mL) and dividing into two equal portions. Afterfiltration, the solutions were sequentially purified by prep HPLC(Waters prep HPLC system; Stationary phase: Waters DeltPak C18 5 □M, 100Angstrom, 300×50 mm I.D., P/N 011801, WAT 011801, No. 330009125W; Mobilephase 90:10 to 50:50 water-acetonitrile with 0.1% formic acid over 30minutes). The combined fractions were concentrated under reducedpressure to an aqueous solution, removing the acetonitrile. The clearaqueous solution was treated with saturated aqueous potassium carbonateto give a cloudy white suspension. The suspension was allowed to standat room temperature overnight. The solid white precipitate that hadformed was collected by vacuum filtration and was dried in the vacuumoven (77° C.) overnight to afford the title compound as a white solid(0.506 g). melting point 157-158° c.

The ¹H NMR and mass spec. analytical data for Examples 1-7 are reportedbelow in Table 1:

TABLE 1 Example ¹H NMR MS 1 ¹H NMR (400 MHz, d6-DMSO) δ MS (APCI) 9.06(d, J = 0.975 Hz, 1H), 8.38 (d, m/z 293.1 J = 5.5 Hz, 1H), 7.98 (m, 1H),7.91 (M + H)⁺ (t, J = 7.8 Hz, 1H), 7.84 (d, J = 1.8 Hz, 1H), 7.66 (s,1H), 7.50 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.2 Hz, 2 ¹H NMR (400 MHz,DMSO-d₆) δ MS (APCI, ppm 9.08 (s, 1H), 8.40 (d, J = 5.5 Hz, M + 1) 307.31H), 8.01 (dd, J = 0.78, 5.7 Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.64 (s,1H), 7.50 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 6.76 (s, 1H),2.34 (s, 3 ¹H NMR (400 MHz, DMSO-d₆) δ MS (APCI) ppm 9.07 (s, 1H), 8.40(d, J = 5.7 Hz, m/z 321.2 1H), 8.00 (dd, J = 0.78, 5.7 Hz, 1H), (M + H)+7.89 (t, J = 7.8 Hz, 1H), 7.65 (s, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.32(d, J = 7.6 Hz, 1H), 6.81 (s, 1H), 2.68 (q, 4 ¹H NMR (400 MHz, DMSO-d₆)δ MS (APCI) ppm 9.12 (s, 1H), 8.42 (dd, J = 5.6, m/z 321.2 1.3 Hz, 1H),8.03 (d, J = 5.5 Hz, 1H), (M + H)+ 7.83 (m, 1H), 7.57 (s, 1H), 7.50 (d,J = 8.0 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 2.53 (q, under DMSO peak, 5¹H-NMR (400 MHz, CDCl₃) δ 8.99 MS (s, 1H), 8.41 (d, 1H, J = 5.5 Hz),(APCI⁺) 7.70-7.66 (m, 2H), 7.36 (d, 1H, m/z 333 J = 8.0 Hz), 7.27 (d,1H, J = 0.5 Hz), (MH⁺). 7.14 (d, 1H, J = 7.6 Hz), 2.89-2.83 (m, 4H),2.56-2.49 (m, 2H), 2.47 (s, 6 ¹H NMR (400 MHz, DMSO-d₆) δ MS (APCI) ppm9.11 (s, 1H), 8.41 (d, J = 5.5 Hz, m/z 321.2 1H), 8.01 (d, J = 5.5 Hz.1H), 7.79 (t, (M + H)+ J = 7.7 Hz. 1H), 7.56 (s, 1H), 7.46 (d, J = 8.2Hz, 1H), 7.14 (d, J = 7.4 Hz, 1H), 2.27 (s, 3H), 2.10 (s, 3H), 2.05 7¹H-NMR (400 MHz, CDCl₃) δ ppm (APCI⁺) 9.06 (s, 1H), 8.44 (d, J = 5.7 Hz,1H), m/z 307 7.73 (d, J = 5.7 Hz, 1H), 7.65 (s, 1H), (M + H)⁺ 7.62 (m,1H), 7.37 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.04 (d, J = 7.6 Hz, 1H),2.27 (s, 3H), 2.18 (s, 3H)

Example 82-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridineStep i.2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-thieno[3,2-c]pyridine

A solution of thieno[3,2-c]pyridine (5.0 g) in 100 mL THF under a N₂atmosphere was cooled to −78° C. (acetone/dry ice). n-Butyllithium (28ml, 1.6M in hexanes, freshly opened bottle) was added dropwise overtwenty minutes, while maintaining the internal temperature at or below−64° C. The mixture was stirred for one hour at −78° C. andtriisopropylborate (freshly opened bottle, 10.2 ml) was subsequentlyadded. The cold bath was removed, allowing the mixture to warm to roomtemperature over 30 minutes. The mixture was stirred at room temperaturefor one hour. A mixture of pinacol (5.9 g) in diethyl ether (20 mL) wasadded to the reaction mixture. After twenty minutes, glacial acetic acid(2.2 mL) was added and a precipitate formed in the reaction mixture. Thereaction mixture was filtered through Celite, rinsing with chloroformuntil the UV-active material was eluted (approximately 2500 mL). Thefiltrate was extracted twice with 5% aqueous sodium hydroxide solution(approximately 500 mL total). The aqueous basic phase stood at roomtemperature overnight. The aqueous layer was cooled in a CH₃CN/dry icebath and was neutralized with 10% aqueous hydrochloric acid whilemaintaining the internal temperature at or below 5° C. The neutralizedaqueous solution was extracted into chloroform ten times. The combinedextracts were dried over anhydrous magnesium sulfate and wereconcentrated under reduced pressure to obtain a solid. This product wascombined with the products of three other reactions performed on thesame scale in a similar manner to afford a combined solid (23.52 g). Thecombined solid was dissolved in approximately 1 L of boiling ethanol.The solution was allowed to cool to room temperature. After about fourhours at room temperature, the solution was stored at 4° C. overnight. Atan-colored material precipitated from solution. The liquid was decantedoff through a filter. The solid was dried in a vacuum oven (50° C.) forone hour to afford the title compound (14.02 g) as a tan solid; ¹H-NMR(400 MHz, CDCl₃ with one drop of D₂O to completely dissolve) δ 1.39(12H, s), 7.89 (1H, d, J=5.3 Hz), 7.97 (1H, s), 8.45 (1H, d, J=4.5 Hz),9.18 (1H, s); MS (APCI⁺) m/z 262.

Alternatively,2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-thieno[3,2-c]pyridinewas also produced as follows:

To a −70° C. solution of thieno[3,2-c]pyridine (21.6 g) in 400 mL of drytetrahydrofuran under nitrogen gas was added n-butyl lithium (99.9 ml ofa 1.6 molar solution in hexanes) over about 15-20 minutes keeping thetemperature below −65° C. The solution became cloudy and dark brown.After 45 minutes tri-isopropyl borate (33.06 g) was added and themixture became homogeneous. The mixture was stirred at −70 to −75° C.for 2 hours. The mixture was allowed to warm to −30° C., then a solutionof pinacol in 100 mL of diethyl ether was added over 4 minutes. Thereaction mixture became an orange homogeneous solution. The temperaturewas allowed to warm to 5° C. and held at that temperature for 1 hour.The reaction mixture was neutralized (pH 7) very slowly with 40 mL of 4Mhydrochloric acid in 1,4-dioxane, keeping the temperature below 10° C. Aprecipitate formed. The reaction mixture was stirred for 1 hour and thenthe solid was collected by filtration. The solid was dried under vacuumat 40° C. for 16 hours. The dry solid (36 g) was stirred with 400 mL ofwater for 1.5 hours. The slurry was filtered and the solid collected.The solid was dried under a flow of air for 60 hours to give the desiredproduct (34.5 g). ¹H NMR (400 MHz, Chloroform-d) δ ppm 1.21 (s, 12H)7.66-7.70 (m, 1H) 7.80 (d, J=0.78 Hz, 1H) 8.28 (d, J=5.65 Hz, 1H) 8.98(d, J=0.97 Hz, 1H).

Step ii:2-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine

An oven-dried 500 mL three-neck round-bottom flask equipped with refluxcondenser and gas inlet valve was charged with2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-thieno[3,2-c]pyridine(11.56 g), trifluoro-methanesulfonic acid5-ethyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl ester (16.4 g),potassium phosphate tribasic (28 g), and 1,4-dioxane (300 mL). Thereaction vessel was evacuated and flushed with nitrogen gas. Thedegassing and nitrogen flush was repeated twice. To the mixture wereadded dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (7.3 g), and 1,1′-bis(diphenylphosphino)ferrocene(5.0 g) and the stirring mixture was degassed and flushed with nitrogengas three more times, and subsequently brought gradually to gentlereflux over a thirty minute period. The mixture was stirred at refluxunder nitrogen for 2.5 hours, and potassium fluoride (13 g) and water(1.2 mL) were subsequently added. The stirring mixture continued atreflux for fifteen minutes and was subsequently cooled slightly andfiltered through Celite. The filter pad was washed with ethyl acetate (1L) and the filtrate was treated with activated carbon. The carbonsuspension was filtered through Celite. This filter pad was washed withadditional ethyl acetate (300 mL), and the filtrate was concentratedunder reduced pressure to afford a crude, red-brown oily residue(approximately 35 g). The residue was treated with several millilitersof absolute ethanol and was concentrated under reduced pressure. Theresidue was mixed with flash silica gel (100 g) and a solution of 5%methanol in ethyl acetate (200 mL). The mixture was filtered and thesilica was washed twice with 200 mL of fresh 5% methanol in ethylacetate solution. The filtrate was concentrated under reduced pressureto afford a red-brown oily residue (approximately 25 g). The residue wasdissolved in several milliliters of ethanol. A precipitate subsequentlyformed upon standing, and was collected by vacuum filtration. The solidswere washed with heptane to afford a sticky, red-orange powder (4.6 g).The ethanol-heptane filtrate yielded up a precipitate that was collectedby vacuum filtration to give a beige solid (4.3 g). The beige solid wasdissolved in absolute ethanol over steam. Gradual cooling of the clearsolution produced a precipitate that was collected by vacuum filtration.Suction drying yielded a solid (2.25 g). The solid was boiled in andprecipitated from absolute ethanol (15 mL) to give a white solid (1.62g). The solid was boiled in and precipitated again from ethanol toprovide the title compound as a white solid (1.20 g). ¹H-NMR (400 MHz,CDCl₃) 9.12 (s, 1H), 8.46 (d, J=5.9 Hz, 1H), 7.94 (d, J=6.0 Hz, 1H),7.68-7.64 (m, 2H), 7.48-7.43 (m, 2H), 7.04 (d, J=7.6 Hz, 1H), 2.21 (s,3H), 2.19 (s, 3H); MS (APCI⁺) m/z 307.

Example 92-[4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridineStep is 2-methyl-6-(4-methyl-pyrazol-1-yl)-pyridine Reaction Condition 1

To a solution of 4-methyl-pyrazole (1.0 g) in 50 mL of anhydrousacetonitrile was added cesium carbonate (5.9 g), followed by2-flouro-6-methylpyridine (1.5 g). The reaction mixture was stirred atreflux for 18 hours. The reaction mixture was filtered to remove cesiumsalts and then the filtrate was evaporated to give a crude oil.Purification by flash chromatography (silica gel, 20% ethyl acetate inheptane) provided, after drying, 1.32 g of the title compound as ayellow oil. ¹H-NMR (400 MHz; DMSO-d₆) δ 8.34 (s, 1H), 7.80 (t, 1H), 7.63(d, 1H), 7.59 (s, 1H), 7.13 (d, 1H), 2.47 (s, 3H), 2.08 (s, 3H); MS(APCI⁺) m/z 174 (MH⁺).

Reaction Condition 2

The reaction carried out for Step i for reaction 1 was carried out in asimilar manner on a larger scale, starting with 4-methyl-pyrazole (5.0g). 5.92 g of the title compound was produced.

Reaction Condition 3

Alternatively: starting with 4-methyl-pyrazole (1.0 g) in acetonitrileusing 1.1 eq of potassium t-butoxide as base, 1.75 g of the titlecompound was produced.

Reaction Condition 4

Alternatively: starting with 4-methyl-pyrazole (1.0 g) in THF using 1.1eq of potassium t-butoxide as base, 0.238 g of the title compound wasproduced.

Reaction Condition 5 In addition, the title compound of Step i wasprepared as follows: To a suspension of 60% sodium hydride (8.04 g) inanhydrous DMF (40 mL) under a nitrogen atmosphere was added dropwise asolution of 4-methyl-pyrazol (15 g) in 30 ml of DMF in an ice bath overa 40 minute period. The resulting mixture was stirred at roomtemperature for 0.5 hour and then 2-flouro-6-methylpyridine (22.33 g)was added dropwise. The mixture was stirred at 80° C. for 3 hours andcooled down. The reaction mixture was poured in ice-water and extractedwith ethyl acetate two times. The combined organic phase was washed withwater and brine, dried over Na₂SO₄ and filtered. The filtrate wasconcentrated in vacuo to give a residue, which was purified bychromatography (2%-10% ethyl acetate in heptanes) to give 29.9 g of thetitle compound as a colorless oil.

Step ii: 2-(5-bromo-4-methylpyrazol-1-yl)-6-methylpyridine

To a cold (−60° C.) solution of2-methyl-6-(4-methyl-pyrazol-1-yl)-pyridine (0.52 g) (prepared byReaction Condition 1 in Step i) in 14 mL of anhydrous THF was added 1.33mL of a 2.5M solution of n-butyllithium (0.21 g) in hexanes. Thereaction mixture was stirred at −60° C. for one hour. A 10 mL solutionof N-bromosuccinimide (0.59 g) in THF was added and then the finalsolution was stirred at −60° C. for 2 hours. Saturated ammonium chloride(20 mL) was added and then the reaction mixture was allowed to warm to24° C. The reaction mixture was diluted with 300 mL of ethyl acetate andthen the aqueous phase was separated. The organic phase was washed withadditional saturated ammonium chloride (50 mL) and then with brine (50mL). The organic layer was separated, dried (sodium sulfate), filtered,and then the filtrate was evaporated to give a crude red oil.Purification by flash chromatography (silica gel, 20% ethyl acetate inheptane) provided, after drying, 0.377 g of the title compound as ayellow oil; ¹H-NMR (400 MHz; DMSO-d₆) δ 7.88 (t, 1H), 7.69 (s, 1H), 7.44(s, 1H), 7.32 (s, 1H), 2.50 (s, 3H), 2.02 (s, 3H); MS (APCI⁺) Mk 254(MH⁺).

Alternative Reaction Conditions

The compound of Step ii,2-(5-bromo-4-methylpyrazol-1-yl)-6-methylpyridine, was also prepared asfollows:

1.) Using similar reaction conditions as described above,2-(5-bromo-4-methylpyrazol-1-yl)-6-methylpyridine, was preparedbeginning with 2-Methyl-6-(4-methyl-pyrazol-1-yl)-pyridine (1.0 g) toprovide the title compound (829 mg).

2.) Using similar reaction conditions as described above,2-(5-bromo-4-methylpyrazol-1-yl)-6-methylpyridine, was preparedbeginning with 2-Methyl-6-(4-methyl-pyrazol-1-yl)-pyridine (5.0 g) toprovide the title compound (2.64 g).

3.) Using similar reaction conditions as described above,2-(5-bromo-4-methylpyrazol-1-yl)-6-methylpyridine, was preparedbeginning with 2-Methyl-6-(4-methyl-pyrazol-1-yl)-pyridine (1.0 g) toprovide the title compound. In addition, 1,2-dibromotetrafluoroethane(1.5 equivalents) was used instead of N-bromosuccinimide (NBS) as thebrominating agent (756 mg).

Step iii:2-[4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine

To a solution of 2-(5-bromo-4-methylpyrazol-1-yl)-6-methylpyridine (2.0g) in 20 mL of anhydrous 1,2-dimethoxyethane (DME) was added palladiumbistriphenylphosphine dichloride (0.153 g). The reaction mixture wasstirred at room temperature for 30 minutes and then 20 mL of water wasadded, followed by sodium bicarbonate (2.0 g) and thentrifluoro-methanesulfonic acid5-ethyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl ester (3.1 g) wasadded. The reaction mixture was refluxed for 18 hours and then cooled toroom temperature. The reaction mixture was diluted with 400 mL ofdiethyl ether and then the organic phase was washed with water (100 mL),saturated sodium bicarbonate (2×100 mL), and brine (100 mL). The organiclayer was dried (magnesium sulfate), filtered, and then the filtrate wasevaporated to give a yellow slurry. The mixture was diluted with 25 mLof ethyl acetate. The material did not all go into solution. The mixturewas triturated for 15 minutes and then filtered (first filtration) toremove a yellow solid, which was rinsed with ethyl acetate (2×10 mL).The solid was air dried for 15 minutes to afford 448 mg of slightlyimpure product. The filtrate from the first filtration above wasevaporated onto 6 g of silica gel and flash chromatographed (silica gel,80% ethyl acetate in heptane, ethyl acetate, followed by 95% ethylacetate in methanol) to give 0.565 g. The combined weight of the titlecompound was 1.01 g as a yellow solid. Precipitation from ethanol gavethe title compound (0.764 g) as a yellow solid; melting point 155-156°C.; MS (APCI⁺) m/z 307 (MH⁺).

Example 102-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridineStep i: 4-Bromo-pyridine

4-Bromopyridine hydrochloride (20.0 g, 102.9 mmol) was partitionedbetween EtOAc and 5% NaHCO₃. The layers were separated, and the aqueousphase extracted with EtOAc. The combined organic extracts were washedwith water and brine, dried over Na₂SO₄, and evaporated under reducedpressure in a 40° C. bath to provide 13.7 g (84%) of a volatile oilwhich was used immediately in the next step.

Step ii: 4-Bromo-pyridine-3-carbaldehyde

A suspension of 4-bromopyridine (13.7 g, 86.8 mmol) in 300 mL THF waspurged with N₂ and cooled to −78° C. under an atmosphere of dry N₂. Asolution of lithium diisopropylamide (2.0 M solution inheptane/THF/ethylbenzene, 43.4 mL, 86.8 mmol) was added over about 3minutes. After 30 minutes, DMF (dimethylformamide) was added. Thereaction mixture was maintained at −78° C. for 3 hours before allowingit to warm to ambient temperature overnight. The reaction mixture wasquenched with saturated NH₄Cl and concentrated under reduced pressure toremove most of the THF. The aqueous mixture was extracted with EtOAc.The combined organic extracts were washed with water and brine, driedover Na₂SO₄, evaporated, and chromatographed (150 g Analogix silica gelcolumn eluting with a gradient of 100% heptane to 30% EtOAc/heptane over45 minutes). Fractions containing the product were combined andevaporated to give 2.20 g (13.6%) of the title compound. MS m/z 186(M+H)⁺.

Step iii: 1-Thieno[3,2-c]pyridin-2-yl-propan-1-one

To a solution of sodium hydrosulfide hydrate (1.31 g, 17.7 mmol) in 5 mLwater was added DMF (30 mL) and K₂CO₃ (3.27 g, 23.7 mmol). The mixturewas cooled to 0° C. in an ice bath before adding 1-bromo-2-butanone(2.68 g, 17.7 mmol). After 15 minutes, a solution of4-bromopyridine-3-carbaldehyde (2.20 g, 11.8 mmol) in DMF (20 mL) wasadded. The reaction mixture was stirred at 45° C. for 2.5 days. Thereaction mixture was diluted with water and extracted with EtOAc. Thecombined organic extracts were washed with water and brine, dried overNa₂SO₄, and evaporated to give the crude product (1.80 g, 80%) which wasused in the next step without further purification. MS m/z 192 (M+H)⁺.

Step iv: 3-Dimethylamino-2-methyl-1-thieno[3,2-c]pyridin-2-yl-propenone

A solution of 1-thieno[3,2-c]pyridin-2-yl-propan-1-one (1.80 g, 9.41mmol) and dimethoxymethyl-dimethyl-amine (5.32 mL, 37.8 mmol) in DMF (20mL) was purged with N₂ and heated to 80° C. for 18 hours. The reactionmixture was diluted with 1:1 water/brine and extracted with EtOAc. Theorganic extracts were washed with 1:1 water/brine and brine, dried overNa₂SO₄, and evaporated to give 2.1 g of crude material. The residue wasrepeatedly dissolved in toluene and evaporated until a constant residueweight was obtained. MS m/z 247 (M+H)⁺.

Step v:2-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine

A solution of3-dimethylamino-2-methyl-1-thieno[3,2-c]pyridin-2-yl-propenone (1.31 g,5.32 mmol) in acetic acid (20 mL) was heated to 90° C. A solution of(6-methyl-pyridin-2-yl)-hydrazine in acetic acid (8 mL) was heated to90° C. before adding to the3-dimethylamino-2-methyl-1-thieno[3,2-c]pyridin-2-yl-propenone solution.The mixture was heated at 95° C. for 15 minutes before removing fromheat and stirring at ambient temperature for 18 hours. The solvent wasremoved under reduced pressure, and the residue was evaporated fromtoluene twice. The residue thus obtained was chromatographed (Analogix110 g silica gel column eluting with 100% CH₃CN) to provide 0.45 g (28%)of an orange oil.

A second method of preparing the compound of Step iii of Example 10,(1-thieno[3,2-c]pyridin-2-yl-propan-1-one) was also carried out asfollows:

Step i. 4-Bromo-pyridine-3-carbaldehyde

A suspension of 4-bromopyridine hydrochloride (5.0 g, 25.7 mmol) in THF(100 mL) was purged with N₂ and cooled to −78° C. A solution of lithiumdiisopropylamide (2.0 M solution in heptane/THF/ethylbenzene, 27.0 mL,54.0 mmol) was added over 3 minutes. After 30 minutes, DMF (8.56 mL,110.6 mmol) was added. The reaction mixture was stirred at −78° C. for15 minutes before rapidly warming to room temperature in a water bath.The reaction was allowed to stir at ambient temperature for 18 hours.The reaction was quenched with saturated NH₄Cl (100 mL) and extractedwith EtOAc. The combined organic extracts were washed with water andbrine, dried over Na₂SO₄, evaporated, and chromatographed (Analogix 150g silica gel column eluting with a gradient of 100% heptane 50%EtOAc/heptane over 1 hour) to give 1.50 g (31%) of the title compound asa light yellow solid. MS m/z 186 (M+H)⁺.

Step ii: Thieno[3,2-c]pyridine-2-carboxylic acid methyl ester

To a solution of 4-bromo-pyridine-3-carbaldehyde (1.50 g, 8.06 mmol) inDMF (10 mL) and water (1 mL) was added K₂CO₃ (1.34 g, 9.68 mmol) andmethyl thioglycolate (0.87 mL, 9.68 mmol). The mixture was heated at 45°C. for 18 hours. The reaction mixture was removed from the heating bathand diluted with water (50 mL). After 1 hour, the fluffy solid thatformed was filtered and washed with water. The material thus obtainedwas dried in a 60° C. vacuum oven to a constant weight of 0.92 g (59%).MS m/z 194 (M+H)⁺.

Step iii. Thieno[3,2-c]pyridine-2-carboxylic acid methoxy-methyl-amide

A suspension of O,N-dimethylhydroxylamine hydrochloride (2.32 g, 23.8mmol) in THF (20 mL) was purged with N₂ and cooled to −78° C. beforeadding n-butyllithium (1.6 M in hexanes, 30.0 mL, 48.1 mmol). Removedbath and stirred 15 minutes before replacing the bath and adding asolution of thieno[3,2-c]pyridine-2-carboxylic acid methyl ester (0.92g, 4.76 mmol) in THF (10 mL). After 45 minutes, saturated NH₄Cl wasadded. The mixture was diluted with EtOAc; washed with water, 1 M HCl,water, 5% NaHCO₃, water, and brine; dried over Na₂SO₄, and evaporated togive 0.54 g (51%) of an oil which solidified upon standing. The productthus obtained was used in the next step without further purification. MSm/z 223 (M+H)⁺.

Step iv. 1-Thieno[3,2-c]pyridin-2-yl-propan-1-one

Thieno[3,2-c]pyridine-2-carboxylic acid methoxy-methyl-amide (0.54 g,2.43 mmol) was dissolved in THF (10 mL), purged with N₂, and cooled to0° C. in an ice bath. Ethyl magnesium bromide (3 M in ether, 2.43 mL,7.29 mmol) was added, and the reaction was allowed to warm to ambienttemperature overnight. The reaction mixture was not purified further. MSm/z 192 (M+H)⁺.

A third second method of preparing the compound of Step iii of Example10, (1-thieno[3,2-c]pyridin-2-yl-propan-1-one) was also carried out asfollows:

Step i: N-methoxy-N-methyl-propionamide

To a stirring mixture of O,N-dimethyl-hydroxylamine hydrochloride (11.1g) and propionyl chloride (9.4 mL) in dichloromethane (300 mL) at 0° C.under a nitrogen atmosphere was added pyridine (18.2 mL). The cold bathwas removed and the mixture was allowed to warm gradually to roomtemperature, at which temperature it stirred over the weekend. A whiteprecipitate had formed in the stirring mixture. The mixture was treatedwith 10% aqueous hydrochloric acid and was stirred until the precipitatehad dissolved in the biphasic solution. The layers were separated andthe dichloromethane phase was washed with saturated aqueous sodiumbicarbonate (100 mL) and brine solution (100 mL), was dried overanhydrous magnesium sulfate, and was concentrated under reduced pressureto afford the title compound as a clear colorless oil (12.05 g); ¹H-NMR(400 MHz; CDCl₃) δ 3.62 (s, 3H), 3.11 (s, 3H), 2.38 (quartet, J=7.6 Hz,2H), 1.07 (t, J=7.6 Hz, 3H); ¹³C-NMR (100 MHz; CDCl₃) δ 175.5, 61.3,32.5, 25.4, 8.9; MS (APCI⁺) m/z 118 (MH⁺).

Step ii: 1-thieno[3,2-c]pyridin-2-yl-propan-1-one

To a stirring mixture of thieno[3,2-c]pyridine (1.0 g) in anhydroustetrahydrofuran (50 mL) at −40° C. (acetonitrile-dry ice bath) was addeda solution of n-butyllithium (1.6 M, 4.7 mL) in hexanes. The mixture wascooled to −78° C. followed by the subsequent addition of a solution ofN-methoxy-N-methyl-propionamide (0.97 g) in anhydrous tetrahydrofuran(10 mL). The cold bath was removed and the reaction mixture was allowedto warm gradually to room temperature. The mixture was stirred overnight(14 hours). The mixture was treated with saturated aqueous ammoniumchloride (150 mL). The mixture was then extracted with ethyl acetate(500 mL), and the extract was washed with brine solution (100 mL). Theorganic phase was dried over anhydrous potassium carbonate and wasconcentrated under reduced pressure, reconstituted in chloroform, andconcentrated again under reduced pressure to afford the orange-brownsemisolid (1.17 g). The semisolid was purified by flash silicachromatography using an Analogix silica column (115 g silica), elutingwith 0-30% ethyl acetate/heptane, then 30-60% ethyl acetate/heptane.Concentration of fractions containing product gave 0.49 g of product.Starting material (0.45 g) was also isolated by evaporation ofappropriate fractions.

Example 112-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine

A 500 mL three-neck round-bottom flask equipped with reflux condenserand gas inlet valve was charged with2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-thieno[3,2-c]pyridine(10 g), trifluoro-methanesulfonic acid4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl ester (14 g),potassium phosphate tribasic (24 g), and 1,4-dioxane (200 mL). Thereaction vessel was evacuated and flushed with nitrogen gas. Thedegassing and nitrogen flush was repeated twice. To the mixture wereadded dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct (3.2 g), 1,1′-bis(diphenylphosphino)ferrocene(2.2 g), potassium fluoride (11 g), and water (1 mL) and the stirringmixture was degassed and flushed with nitrogen gas three more times, andsubsequently brought gradually to 80° C. over a twenty minute period.The mixture was stirred at 80° C. for ten minutes and was subsequentlycooled gradually to room temperature as it stood overnight. The mixturewas vacuum filtered through Celite and was washed through the filterplug with two portions of ethyl acetate (800 mL and 600 mL). Thefiltrates were combined and concentrated under reduced pressure to givea dark residue. The residue was dissolved in dichloromethane (200 mL). Asolid precipitated and was removed by vacuum filtration. The filtratewas concentrated under reduced pressure to afford an orange-brown solid(20 g). The solid was treated with room-temperature acetonitrile (200mL) and the resulting suspension was swirled and vacuum filtered toafford a yellow solid (9.1 g). This second solid was dissolved inboiling acetonitrile over a steam bath (75 mL solution volume hot) andthe orange solution was allowed to cool to room temperature over thirtyminutes. A solid precipitated, and the flask was sealed with and storedat 4° C. overnight. The precipitate was vacuum filtered and suctiondried to afford the title compound as an off-white solid (4.80 g). Thissolid was combined with batches of solid isolated in a similar mannerfrom other experiments (21.5 g total mass) to give a combined solid.

The combined solid was further combined with the solid (8.92 g) obtainedfrom the following experiment:

Solutions of3-dimethylamino-2-methyl-1-thieno[3,2-c]pyridin-2-yl-propenone (31.35 g)in glacial acetic acid (500 mL) and (6-methyl-pyridin-2-yl)-hydrazine(15.67 g) in glacial acetic acid (200 mL) were purged with dry nitrogenand heated to 90° C. under nitrogen gas. The two mixtures were combinedand heated to 95° C. for 15 minutes. Heating was discontinued and thereaction mixture was allowed to cool and stir at room temperatureovernight. The crude reaction mixture was concentrated to about 100 g ofa dark oil under reduced pressure. The residue was dissolved in tolueneand evaporated to about 70 g. A strong smell of acetic acid was evident,so the evaporate was dissolved in toluene and evaporated to about 70 gof an oil. Although the residue thus obtained still had a faint aceticacid odor, it was dissolved in acetonitrile (200 mL) and allowed tostand at room temperature. After 2.5 days, no precipitate had formed, sothe solution was evaporated under reduced pressure in a 60° C. bath. Theresulting oil was dissolved in acetonitrile (about 200 mL) and allowedto cool to room temperature. A small amount of precipitate had formedwhich was collected and washed with a small amount of acetonitrile. Uponfiltration, a large amount of solid precipitated from the mother liquor.The precipitate was collected and washed with a small amount ofacetonitrile. Upon drying in a 60° C. vacuum oven, 12.05 g of materialwas obtained. The solid thus obtained was reisolated from 250 mL boilingacetonitrile to provide, after drying to constant weight in a vacuumoven at 60° C., the solid (8.92 g)2-[4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine.

The combined 21.5 g and 8.92 g batches were suspended in acetonitrile to500 mL total volume. The suspension was heated over a steam bath toboiling. Most of the solid dissolved into solution, but someparticulates remained undissolved and were removed by hot vacuumfiltration. The filtrate collected in the 2 L filter flask rapidlyyielded a white solid precipitate. Acetonitrile was added to 600 mLvolume. The filtrate was boiled over a steam bath until all solidsdissolved. The flask was removed from the steam bath and the mixture wascooled gradually to room temperature as it stood on the bench for 2.5hours. A white solid formed from solution over that time and wascollected by vacuum filtration to afford the title compound as a whitesolid (24.85 g); ¹H-NMR (400 MHz, CDCl₃) & 9.06 (d, J=0.8 Hz, 1H), 8.43(d, J=5.7 Hz, 1H), 7.72 (ddd, J=5.7, 1.0, 0.8 Hz, 1H), 7.64 (s, 1H),7.61 (t, J=7.8 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.31 (d, J=0.8 Hz, 1H),7.03 (d, J=7.6 Hz, 1H), 2.26 (s, 3H), 2.18 (s, 3H); MS (APCI⁺) m/z 307(MH⁺), (APCI) m/z 305 (M−H⁺).

Example 122-(4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[2,3-c]pyridine

Thieno[2,3-c]pyridine of Step iii, Example 12 has been synthesizedpreviously (see e.g., Graulich at al. (2004) Synthesis 12: 1935-1937;Graulich et al. (2005) J. Med. Chem. 48(15): 4972-4982).

Step i. (2,2-Dimethoxy-ethyl)-thiophen-2-ylmethylene-amine

A solution of thiophene-2-carboxaldehyde (7.5 mL, 82.3 mmol, AlfaAesar), aminoacetaldehyde dimethyl acetal (8.82 mL, 82.3 mmol, AlfaAesar) in 100 mL toluene was heated to 115° C. using a Dean-Stark trapto remove water. After 3 hours, the reaction was cooled to roomtemperature. The reaction was placed on a rotary evaporator to evaporatethe toluene, which yielded a brown liquid (about 17.3 g), which was usedwithout further manipulation in the next step. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 3.42 (6H, s), 3.73 (1H, d, J=5.3 Hz), 4.65 (1H, t,J=5.3 Hz), 7.07-7.41 (3H, m).

Step ii.{[(2,2-Dimethoxy-ethyl)-ethoxycarbonyl-amino]-thiophen-2-yl-methyl}-phosphonicacid dimethyl ester

To the oil obtained in Step i (16.3 g, 82.3 mmol) was added 60 mL THFunder a N₂ atmosphere. The reaction was cooled to −10° C. (MeOH/ice) andfitted with an internal thermometer. Ethyl chloroformate (7.9 mL, 82.3mmol) was added dropwise keeping the internal temperature at or below−9.5° C. The reaction was stirred for 10 minutes at −10° C. and then thebath was removed and the reaction was allowed to warm to roomtemperature. Using an ice bath as a heat sink, trimethyl phosphite (10.7mL, 90.5 mmol) was added. The reaction was allowed to gradually warm toroom temperature and was stirred overnight. The solvent was removed on arotary evaporator. Toluene was added and evaporated on a rotaryevaporator. The toluene addition and evaporation was then carried out asecond time.

The resulting thick brown oil was purified by column chromatography(Biotage Horizon system, 330 g Isco RediSep column, 0-100% EtOAc/hexaneover 1 column volume then hold at 100% EtOAc). The desired fractionswere combined and concentrated to give 23.48 g of a thick, brown oil.

Step iii. Thieno[2,3-c]pyridine

The material from Step ii (23.5 g, 61.6 mmol), was dissolved in 100 mLCH₂Cl₂. The three neck flask was fitted with a reflux condenser andinternal thermometer under N₂ atmosphere. The flask was evacuated andpurged with nitrogen. Titanium (IV) chloride (40 mL, 369 mmol) was addedto the reaction slowly. The reaction temperature was maintained ataround 40° C. After about 15 mL were added, the reaction was placed inan ice bath to control the temperature. The reaction was allowed togradually warm to room temperature before heating to 40° C. overnight.The reaction was heated to 40° C. for 18 hours, then cooled to roomtemperature. The reaction contents were poured, in portions, into alarge beaker containing 200 g of ice and 200 mL NH₄OH with lots offuming observed. The reaction was stirred vigorously for a few minutes.The reaction was filtered and the solid was rinsed with CHCl₃ (3×100mL).

The biphasic filtrate was transferred to a separatory funnel and thelayers were separated. The organic layer was extracted into 1 N HCl(2×100 mL). The aqueous HCl layer was washed with 20 mL CH₂Cl₂ andcarefully basified with concentrated NH₄OH until a pH of about 9 wasreached. The product was extracted into CH₂Cl₂ (3×100). The organiclayer was dried over MgSO₄, filtered, concentrated to an orange oil thatsolidified to an orange solid. The crude material was purified by columnchromatography (Biotage Horizon system, 120 g Isco RediSep column,equilibrate with 100% heptane, elute with 45% EtOAc/heptane). A whitesolid was isolated (4.20 g). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.56 (1H,dd, J=5.5, 0.8 Hz), 7.86 (1H, dd, J=5.5, 1.0 Hz), 8.10 (1H, d, J=5.3Hz), 8.46 (1H, d, J=5.5 Hz), 9.25 (1H, s). ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 7.40 (1H, d, J=5.5 Hz), 7.74 (2H, m), 8.52 (1H, d,J=5.5 Hz), 9.19 (1H, s). MS (APCI, M+1) 136.0.

Step iv. Thieno[2,3-c]pyridin-2-ylboronic acid trihydrogen phosphate

A three neck flask fitted with an internal thermometer containing thematerial from Step iii (4.12 g, 30.5 mmol) was evacuated and then filledwith nitrogen atmosphere. THF (50 mL) was added and the solution cooledto −44° C. (CH₃CN/dry ice). n-Butyllithium (1.6M/hexane, 21 mL, 34 mmol)was added over 10 minutes, while maintaining the internal temperature ator below −35° C. The reaction was stirred at −33 to −45° C. for 75minutes. Triisopropyl borate (8.4 mL, 36.6 mmol) was added and thecooling bath removed. The reaction was stirred for one hour thenphosphoric acid (85% aqueous, 2.5 mL, 33.5 mmol) was added. The reactionwas diluted with 10 mL water resulting in a precipitate. The reactionwas stirred vigorously for 15 minutes. A yellow solid was collected byfiltration, rinsing with EtOAc (about 50 mL) to provide a white powderysolid (9.57 g). Approximately half of the material was washed with 50 mLwater to isolate 3.16 g of a pale pink solid. Both crops of material arethe same as determined by spectral data. ¹H NMR (400 MHz, METHANOL-d₄) δppm 7.87 (1H, d, J=5.5 Hz), 7.95 (1H, s), 8.41 (1H, d, J=5.7 Hz), 9.11(1H, s).

Step v.2-(4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[2,3-c]pyridine

In a 250 mL three neck flask was added trifluoro-methanesulfonic acid4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl ester (1.23 g, whichmay be synthesized as describe in Example 7, Step iv), the material fromStep iv (2.00 g), potassium fluoride (1.11 g), potassium phosphatetribasic (2.44 g) in 50 mL dioxane. The flask was evacuated with avacuum and purged with nitrogen gas. To this was added PdCl₂(dppf)(Strem, Dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct, 280 mg)) and dppf ligand (Strem,1,1′-bis(diphenylphosphino)ferrocene, 212 mg) and water (0.10 mL). Thereaction was heated to 80-111° C. for 2.5 hours then cooled to roomtemperature. The reaction was combined with material from anotherreaction which was run in a similar manner. The crude reaction materialwas filtered through a pad of Celite rinsing with EtOAc (about 750 mL).The filtrate was concentrated to dryness on rotary evaporator. The darkresidue was diluted with CH₂Cl₂ and the solid was filtered off. TheCH₂Cl₂ filtrate was concentrated on a rotary evaporator to a blackresidue. The crude material was purified by filtering through a silicagel plug eluting with 50% EtOAc/hexane (about 800 mL total). Thefractions from the silica gel plug filtration yielded two lots ofmaterial (one pure, one impure) These were concentrated to drynessseparately. The impure material was triturated with Et₂O and a whitesolid was collected by filtration. The filtrate was concentrated and theEt₂O trituration was repeated to give 83 mg. The fractions containingonly desired product plus the 83 mg batch were combined and purifiedtogether with the pure lot. About 5-10 mL of EtOH were added to thematerial, which was heated to boiling. Once all of the material wasdissolved, it was removed from the heat and allowed to stand. A beigesolid then began to precipitate. The mother liquors were decanted offand the precipitate was rinsed with a small amount of cold EtOH. Theprecipitate was dried in a 50° C. vacuum oven overnight to provide 550mg of a beige solid. ¹H NMR (400 MHz, CDCl₃) δ ppm 9.09 (1H, s), 8.50(1H, d, J=5.7 Hz), 7.62-7.69 (3H, m), 7.39 (1H, d, 7.99 Hz), 7.27 (1H,d, J=0.6 Hz), 7.04 (1H, d, J=7.4 Hz), 2.25 (3H, s), 2.19 (3H, s). MS(APCI, M+1) 307.1. mp 124-125° C.

Example 132-(2-(6-methylpyridin-2-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thieno[2,3-c]pyridine

In a 250 mL three neck flask was added trifluoro-methanesulfonic acid2-(6-methyl-pyridin-2-yl)-2,4,5,6-tetrahydro-cyclopentapyrazol-3-ylester (1.33 g, which may be synthesized as described in Example 5, Stepiv), the material from Example 12, Step iv (2.00 g), potassium fluoride(1.11 g), potassium phosphate tribasic (2.44 g) in 45 mL dioxane. Theflask was evacuated under reduced pressure and purged with nitrogen gas.To this was added PdCl₂(dppf) (Strem,Dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium (II)dichloromethane adduct, 280 mg)) and dppf ligand (Strem,1,1′-bis(diphenylphosphino)ferrocene, 212 mg) and water (0.10 mL). Thereaction was heated to 80-111° C. for 3.5 hours and then cooled to roomtemperature. The reaction material was filtered through a pad of Celiterinsing with EtOAc (about 250 mL). The EtOAc filtrate was concentratedto dryness on a rotary evaporator. The resulting dark residue wasdiluted with CH₂Cl₂ and the solid was filtered off. The CH₂Cl₂ filtratewas concentrated on a rotary evaporator to a black residue that wasdiluted with CH₃CN and sonicated. A tan solid (0.520 g) was collected byfiltration. The filtrate was concentrated to dryness and purified bycolumn chromatography (Biotage Horizon system, 12 g Analogix column,0-50% EtOAc/heptane, then hold at 50% EtOAc/heptane) to yield about 300mg of a solid. The 300 mg of the solid and the tan solid (0.520 g) werecombined and diluted with about 10 mL EtOH, heated to boiling, filtered,and allowed to cool to room temperature. Solid material began toprecipitate. After standing overnight at room temperature the motherliquors were decanted off and the remaining material was rinsed with asmall amount of cold EtOH. The solid was dried in a 45° C. vacuum ovenfor 1.5 hours to provide 410 mg of a solid. ¹H NMR (400 MHz, CDCl₃)

ppm 9.05 (1H, s), 8.45 (1H, d, J=5.7 Hz), 7.70 (1H, t, J=7.7 Hz), 7.62(1H, d, J=5.7 Hz), 7.38 (1H, d, J=7.9 Hz), 7.25 (1H, d, J=1.2 Hz), 7.15(1H, d, J 7.6 Hz), 2.85-2.91 (4H, m), 2.51-2.58 (2H, m), 2.46 (3H, s).MS (APCI, M+1) 333.1. mp 145-146° C.

Example 142-[4-methyl-2-(6-methylpyridin-2-yl)2H-pyrazolo-3-yl]thieno[3,2-c]pyridine

Trifluoro-methanesulfonic acid4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl ester (36.6 g, 114mmoL), potassium carbonate (39.4 g, 285 mmol), and2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-thieno[3,2-c]pyridine(31.2 g, 120 mmol) were slurried in toluene (300 mL), isopropyl alcohol(IPA) (75 mL) and water (75 mL). The reaction mixture was sequentiallysubjected to vacuum followed by purging with nitrogen. This was repeatedfor a total of five times.

Bis(triphenylphosphine)palladium(II) Chloride (4.4 g, 6.27 mmol) wasadded, and the reaction was heated to 78° C. After 2 hours the reactionmixture was allowed to cool. The aqueous layer was separated, and theorganic layer was filtered to remove black catalyst residue. The organiclayer was washed with water (80 mL). The organic layer was extractedwith 3 M HCl (100 mL) and water (100 mL). Carbon (0.5 g) and celite (9g) were added to the aqueous and the solution was concentrated to removethe residual toluene. The aqueous layer was filtered. The aqueous wasmade basic (pH=10) with the addition of 50% NaOH. A yellow solidprecipitated. The solid was filtered, washed with water and dried bypulling air through it to give 24.1 g (69.1%) of a yellow solid. Thesolid was slurried in toluene (250 mL). The slurry was heated to 65° C.and filtered. The filtrate was concentrated. The residue was slurried intoluene (100 mL) and filtered. The cake was washed with toluene (40 mL).The cake was dried by pulling air through it to give 19.2 g (55%) ofcrude product.

The 19.2 g was combined with other similarly prepared samples to give141.7 g. The combined solids were slurried in IPA (500 mL) and theslurry was heated to 83° C., but the solid did not all go into solution.IPA (300 mL) was added and the slurry heated to 83° C. to give a darksolution. The solution was allowed to cool to room temperature at 8°C./hour. The slurry was filtered, and the cake was washed with IPA (200ml). The cake was dried at 50° C. for 60 hours to give 131.8 g of thedesired product.

Biological Example 1

ALK-5 kinase assay methods have been described in the art (see e.g.,Laping et al. (2002) Mol. Pharmacol. 2002; 62: 58-62). The compoundsnamed in the specified Examples were tested as follows for inhibition ofALK-5 autophosphorylation activity and of the ALK-5 phosphorylation ofα-Casein.

Materials:

-   -   Buffer. 50 mM HEPES, pH 7.6, with 10 mM NaCl, 10 mM MgCl₂, and 1        mM DTT.    -   GST-ALK-5 protein—0.44 mg/ml (roughly 7 μM stock). A 1:350        dilution gives a 20 nM stock, which translates to 2 nM final in        assay. Human ALK-5 was expressed in Sf9 insect cells infected        with Baculovirus expressing a ALK-5 truncation sequence (amino        acids H¹⁴⁹-M⁵⁰³), fused at the N-terminus to Glutathione        S-transferase GST, in a pFastBac vector (Invitrogen). The cells        were disrupted by sonication at 4° C. The lysate was centrifuged        at 40,000×g for 45 minutes, and the supernatant applied to a 10        ml column of Glutathione Sepharose 4 Fast Flow (Amersham        Bioscienses) equilibrated with 100 mM Tris-HCl pH 7.6 buffer        containing 300 mM NaCl, 10% glycerol, 1% NP40, 2 mM        dithiothreitol (DTT) and one Protease Inhibitor complete        EDTA-free tablet per 50 ml (Roche). The column was washed with 5        column volumes of 50 mM Tris HCl pH 8.0 containing 150 mN NaCl,        10% glycerol, 2 mM DTT and one Protease Inhibitor complete        EDTA-free tablet per 100 ml. The column was eluted with wash        buffer containing 8 mM reduced glutathione. Fractions were        collected and dialyzed overnight in 20 mM Tris HCl pH 8.0        containing 10% glycerol, 150 mM NaCl, 2 mM DTT and 1 mM        4-(2-aminoethyl)-bezenesulfonylfluoride.HCl (AEBSF) (Sigma) at        4° C.    -   α-Casein (Sigma, #C8032) is made up at 2 mM in Buffer (50        mg/ml).    -   Cold ATP contains 10 μM cold ATP (from a 10 mM stock in Buffer).    -   Hot ATP consists of 0.5 μCi/well □-³³P-ATP (Amersham, AH9968) in        Buffer.

Assay Buffer—Per 10 ml Buffer

-   -   1 ml of 500 mM HEPES (pH 7.6)    -   20 μl of 5 M NaCl    -   100 μl of 1 M MgCl₂    -   10 μl of 1 M DTT (dithiothreitol)

Assay Method:

In a 96 well filter-bottom plate (Millipore, #MSDV N6B 50), 58 μl AssayBuffer is added to reach well. Add 10 μl of Cold ATP mix in AssayBuffer, then 10 μl of a 1:10 dilution of α-Casein stock. Then add 2 μlof compound being tested (DMSO) at a 50× final concentration. Hot ATPmix (10 μl) is added, and the reaction is started with the addition of10 μl of a 1:350 dilution of the ALK-5 protein (2 nM final) in AssayBuffer with 0.05% BSA (Bovine Serum Albumin). The reaction is mixed for5 minutes at room temperature, and then continued for 145 minutes atroom temperature. The reaction is then stopped with the addition of 100μl of ice-cold 20% TCA (trichloroacetic acid). The assay is thenincubated for at least 1 hour at 4° C., and then the contents of eachwell are filtered by suction through the filter. The wells are washedthree times with 200 μl ice-cold 10% TCA. The plate bottom is blottedbefore and after removing plastic sub-base, and dried overnight at roomtemperature. Add 30 μl of scintillation fluid, and count 1 minute perwell on a Wallac Tri-Lux scintillation counter.

The IC₅₀ (nM) values are reported in Table 2 below as the mean of two ormore IC₅₀ values that were determined in one or more experiments. Thenumber of determinations (“n”) is reported within the parentheses. Theindividual values that produced the mean IC₅₀ values are listed insidethe parentheses if there were 4 or fewer determinations. The StandardError (SE) is reported as well. The Standard Error is the standarddeviation divided by the number of determinations (“n”).

TABLE 2 IC₅₀ Example (nM) 1 23.4 (n = 18); SE = 3.5 2 94.1 (n = 2; 111,76.8); SE = 17.3 3  108 (n = 2; 121, 94.8); SE = 13.3 4 4.33 (n = 4; 10,2.84, 7.82); SE = 1.51 5 12.9 (n = 4; 31, 12.5, 1.0, 7.24); SE = 6.46 635.2 (n = 2; 38.6, 31.8); SE = 3.41 7 6.65 (n = 16); SE = 0.61 12 7.94(n = 2; 10.4, 5.47); SE = 2.48 13 18.9 (n = 2; 26.7, 11.2); SE = 7.73

Biological Example 2

ALK-5 gene reporter assay methods have been described in the art (seee.g., Maliekal et al. (2004) J Biol Chem 279(35):36287-36292). Thecompounds named in the specified Examples were tested as follows forinhibition of Smad binding element (SBE) luciferase reporter activity inTGFβ1 stimulated NIH-3T3 cells. The following luciferase assay employsNIH/3T3 (murine fibroblast) cells, which are transiently transfectedwith a Smad binding element (SBE) luciferase reporter construct. Thisexpressed construct is responsive to agents that stimulate the Smadsignaling pathway.

Materials:

-   -   NIH-3T3 cells (ATCC CRL-1658)    -   Dulbecco's Modified Eagle Medium with phenol red (Life        Technologies 11965-092)    -   Dulbecco's Modified Eagle Medium without phenol red (Life        Technologies 21063-029)    -   Fetal Bovine Serum (Life Technologies SH30071.03)    -   Fugene (Roche 1814443)    -   Opti-MEM I (Life Technologies 31985-070)    -   Dual-Glo Luciferase Assay System (Promega E2940, E2980)    -   Gentamycin solution (10 milligrams/millimeter)(Life Technologies        15710-064)    -   96-Well Assay Plates, white, TCT (Corning Costar 3917)    -   75 centimeter Tissue Culture Flasks (Corning Costar 430641)    -   pRL-CMV vector (Promega Corporation, Madison, Wis., Product        E2261) (pRL is a vector encoding a Renilla reniformis luciferase        under the control of a constitutively active        cytomegalovirus (CMV) promoter)    -   Transforming Growth Factor □₁-(R&D systems, Minneapolis, Minn.,        Product 240-B)    -   pSBE4-LuGBV4 vector (also known as pSBE4-luc vector)(which        contains 4 copies of a Smad Binding Element and firefly        luciferase coding region) (Zawel et al. (1998) Mol Cell.        1(4):611-617).

Methods:

The NIH-3T3 cells are maintained in Dulbecco's Modified Eagle Mediumwith 10% Fetal Bovine Serum and 10 micrograms/ml Gentamycin. Cells aresplit every Monday, 1:5 to 1:10, and are split again on Wednesday. Cellsare split on Friday, 1:20, for cells required for assay and maintenanceon Monday. Do not let cells grow to total confluency.

Day 1, 0 Hours (Start of Experiment)

Plate 1.6 million NIH/3T3 cells in a 75 centimeter flask fortransfections using 15 milimeters of growth media (Dulbecco's ModifiedEagle Medium, 10% Fetal Bovine Serum, 10 micrograms/millimeterGentamycin).

Day 1, 7 Hours Post-Start of Experiment

Prepare the transfection:

a. In a 1.5 millimeter microcentrifuge tube, add 48 microliters ofFugene directly to 400 microliters of Opti-MEM. Incubate at roomtemperature for 5 minutes.

b. During the above incubation period, aliquot 8 micrograms of SBE-lucand 0.16 micrograms of CMV-pRL to 400 microliters Opti-MEM.

c. Add Fugene/Opti-MEM from step “a” to DNAs in step “b”. Incubate atroom temperature for 15 to 45 minutes.

d. Add complexed DNAs to cells plated above (Day 1, 0 hours). It is notnecessary to change media. Incubate overnight at 37° C., 5% CO₂.

Day 2, 24 Hours Post-Start of Experiment

Prepare cell suspension(s) from transfected cells (Day 1, step 2) at adensity of 200,000 cells/ml in Dulbecco's Modified Eagle Medium, 10%Fetal Bovine Serum, 10 micrograms/millimeter Gentamycin. Plate cells inassay plates (white TCT), 100 microliters/well for 96-well plates. Thiswill put the number of cells at 20,000 cells/well. Incubate for 5-6hours at 37° C., 5% CO₂.

Day 2, 31 Hours Post-Start of Experiment

a. Prepare dose response plates if needed.

b. Wash plate with 100 microliters Dulbecco's Modified Eagle Medium, 10micrograms/millimeter Gentamycin without serum.

c. Add 170 microliters/well of Dulbecco's Modified Eagle Medium, 10micrograms/millimeter Gentamycin, 0.5% Fetal Bovine Serum,

d. Transfer test compounds and controls to assay plates (white, TCT), 20microliters of 10× stock for 96-well plates.

e. After 30-60 minutes of treatment with compound, add 250picograms/millimeter of Transforming Growth Factor D₁ to each well. (Add10 microliters of a 20× stock).

Day 3, 51 Hours Post-Start of Experiment

Assay Plates for Luciferase Activity.

a. Reconstitute lyophilized Dual-Glo Luciferase Substrate with Dual-GloLuciferase Buffer according to manufacturer's directions.

b. Remove assay plates from the incubator and allow them to reach roomtemperature for 10 minutes.

c. Aspirate media from the assay plate. Add 80 microliters dilutedSteady-Glo Luciferase Substrate containing 1 parts Dulbecco's ModifiedEagle Medium media without phenol red. Seal plates and incubate for atleast 10 minutes at room temperature.

d. Read on the Packard TopCount HTS plate reader using single photoncounting (SPC) mode for 6 seconds/well to read the firely luciferaseactivity from pSBE4-Luc/BV4 pSBE4-luc.

e. After reading plates for firefly luciferase activity, remove sealsand add 40 microliters of Stop and Glo reagent to each well. Resealplates and incubate for at least 10 minutes at room temperature. Readplates on Packard TopCount as with firefly luciferase, to read theRenilla luciferase activity from pRL.

The Renilla luciferase activity serves a transfection control. Thefirefly luciferase activity serves as the assay readout. The luciferaseassay activity is normalized to the Renilla assay activity for eachparticular sample.

The IC₅₀ (nM) values are reported in Table 3 below as the mean of two ormore IC₅₀ values that were determined in one or more experiments. Thenumber of determinations (“n”) is reported within the parentheses. Theindividual values that produced the mean IC₅₀ values are listed insidethe parentheses where n is 4 or less. The Standard Error (SE) isreported as well. The Standard Error is the standard deviation dividedby the number of determinations (“n”).

TABLE 3 IC₅₀ Example (nM) 1 149 (n = 4; 117, 133, 164, 183); SE = 14.9 2440 (n = 3; 211, 504, 605); SE = 118 3 403 (n = 3; 286, 453, 471); SE =58.9 4 21.5 (n = 3; 11.5, 26, 27); SE = 5.0 5 30.8 (n = 4; 22, 27, 30,44); SE = 4.71 6 244 (n = 3; 186, 199, 348); SE = 52.0 7 35.9 (n = 21);SE = 2.47 12 42.5 (n = 2; 51.0, 34.0); SE = 8.5 13 57.5 (n = 2; 52.0,63.0); SE = 5.5

Formulation Example 1 Preparation of a gel containing 2% (w/w)2-[4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridineMaterials:

-   -   Ethanol (200 Proof), USP (Aaper Alcohol and Chemical Co, KY)    -   Propylene glycol (Purity >99.5%), ACS reagent (Sigma-Aldrich        Chemicals, St. Louis, Mo.)    -   Polyethylene glycol (PEG 400), Molecular Weight: 380-420        (Mallinkrodt Baker Inc., Phillipsburg, N.J.)    -   Hydroxypropyl cellulose (KLUCEL® HF) (Hercules Incorporated,        Wilmington, Del.)    -   Water, Chromosolve® for HPLC (Sigma-Aldrich Chemicals, St.        Louis, Mo.)    -   Benzyl alcohol (Purity >99%) (Sigma-Aldrich Chemicals, St.        Louis, Mo.)

Procedure:

2-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine(2 g) was transferred into a 150-mL glass bottle. Then propylene glycol(30 g), polyethylene glycol (30 g), water (10 g), and benzyl alcohol (2g) and ethanol (20 g) were added to the bottle. The mixture was stirredfor 2 hours. Then KLUCEL® HF (500 mg) was added to the solution,followed by ethanol q.s. to 100 g. The solution was stirred overnight toyield a gel containing 2% (w/w)2-[4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine.

Formulation Example 2

Preparation of a gel containing 1% (w/w)2-[4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine.

Materials:

-   -   Ethanol (200 Proof), USP (Aaper Alcohol and Chemical Co, KY)        Propylene glycol (Purity >99.5%), ACS reagent (Sigma-Aldrich        Chemicals, St. Louis, Mo.)    -   Polyethylene glycol (PEG 400), Molecular Weight: 380-420        (Mallinkrodt Baker Inc., Phillipsburg, N.J.)    -   Hydroxypropyl cellulose (KLUCEL® HF) (Hercules Incorporated,        Wilmington, Del.)    -   Water, Chromosolve® for HPLC (Sigma-Aldrich Chemicals, St.        Louis, Mo.)    -   Benzyl alcohol (Purity >99%) (Sigma-Aldrich Chemicals, St.        Louis, Mo.)

Procedure:

2-[4-Methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine(1 g) was transferred into a 150-mL glass bottle. Then propylene glycol(30 g), polyethylene glycol (30 g), water (10 g), and benzyl alcohol (2g) and ethanol (20 g) were added to the bottle. The mixture was stirredfor 30 minutes to 1 hour. Then KLUCEL® HF (500 mg) was added to thesolution, followed by ethanol q.s. to 100 g. The solution was stirredovernight to yield a gel containing 1% (w/w)2-[4-methyl-2-(6-methyl-pyridin-2-yl)-2H-pyrazol-3-yl]-thieno[3,2-c]pyridine.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be apparent to persons skilled in the art and areto be included within the spirit and purview of this application and thescope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein: R¹ is athieno[3,2-c]pyridinyl, a thieno[3,2-b]pyridinyl, athieno[2,3-c]pyridinyl, or a thieno[2,3-b]pyridinyl, each of which maybe optionally substituted with one to three substituents eachindependently selected from the group consisting of: C₁-C₃-alkyl,—(C₁-C₃-alkyl)-S—(C₁-C₃-alkyl), —S—C₁-C₃-alkyl,—(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl, —C(O)O—C₁-C₃-alkyl,—C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, wherein R³⁰ and R³¹ are eachindependently selected from the group consisting of: H, and C₁-C₃alkyl-OH, C₁-C₃-alkyl, halo, and —O—C₁-C₃-alkyl; R² and R³ areindependently selected from the group consisting of: hydrogen,C₁-C₃-alkyl, —(C₁-C₃-alkyl)-S—(C₁-C₃-alkyl), —S—C₁-C₃-alkyl,—(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl, —C(O)O—C₁-C₃-alkyl,—C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, and a C₃-C₆-cycloalkyl, whereinR³⁰ and R³¹ are each independently selected from the group consistingof: H, and C₁-C₃ alkyl; or R² and R³ may be taken together to form a 5or 6-membered heteroaryl, a phenyl, a C₄-C₆-cycloalkyl, or a4-6-membered heterocycloalkyl, wherein said C₄-C₆-cycloalkyl or4-6-membered heterocycloalkyl may be optionally substituted with one tothree substituents independently selected from halo, —OH, oxo, and C₁-C₃alkyl, wherein said 5 or 6-membered heteroaryl, or phenyl may beoptionally substituted with one to three substituents independentlyselected from halo, —CN, —OH, —O—C₁-C₃ alkyl and C₁-C₃ alkyl; and R⁴,R⁵, R⁶, and R⁷ are selected from the group consisting of H, —OH,C₃-cycloalkyl, C₁-C₃-alkyl, —(C₁-C₃-alkyl)-S—(C₁-C₃-alkyl),—S—C₁-C₃-alkyl, —(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl,—C(O)O—C₁-C₃-alkyl, —C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, wherein R³⁰and R³¹ are each independently selected from the group consisting of: H,and C₁-C₃ alkyl C1-C₃-alkyl, —O—C₁-C₃-alkyl, and halo.
 2. The compoundof claim 1, or a pharmaceutically acceptable salt thereof, wherein R²and R³ are taken together to form a C₄-C₆-cycloalkyl, or a 4-6-memberedheterocycloalkyl, wherein said C₄-C₆-cycloalkyl or 4-6-memberedheterocycloalkyl may be optionally substituted with one to threesubstituents independently selected from oxo and C₁-C₃ alkyl.
 3. Thecompound of claim 2, or a pharmaceutically acceptable salt thereof,wherein R² and R³ are taken together to form a C₅-cycloalkyl, or a4-6-membered heterocycloalkyl, wherein said 4-6-memberedheterocycloalkyl is selected from the group consisting of: atetrahydrofuranyl, a tetrahydrothienyl, a imidazolidinyl, anoxazolidinyl, an imidazolinyl, an isoxazolidinyl, and a pyrrolidinyl. 4.The compound of claim 3, or a pharmaceutically acceptable salt thereofwherein R¹ is a thieno[3,2-c]pyridinyl or a thieno[2,3-c]pyridinyl,which may be optionally substituted with one to three substituents eachindependently selected from the group consisting of: —OH, C₁-C₃ alkyl,halo, and —O—C₁-C₃ alkyl.
 5. The compound of claim 4, or apharmaceutically acceptable salt thereof, wherein said compound is2-(2-(6-methylpyridin-2-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thieno[3,2-c]pyridine,or a pharmaceutically acceptable salt thereof.
 6. The compound of claim4, or a pharmaceutically acceptable salt thereof, wherein said compoundis2-(2-(6-methylpyridin-2-yl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazol-3-yl)thieno[2,3-c]pyridine,or a pharmaceutically acceptable salt thereof.
 7. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein R² and R³ areindependently selected from the group consisting of hydrogen,C₁-C₃-alkyl, —(C₁-C₃-alkyl)-O—(C₁-C₃-alkyl), —O—C₁-C₃-alkyl,—C(O)O—C₁-C₃-alkyl, —C(O)O—H, —C(O)NR³⁰R³¹, halo, —CN, —OH, and aC₃-C₈-cycloalkyl, wherein R³⁰ and R³¹ are each independently selectedfrom the group consisting of: H, and C₁-C₃ alkyl.
 8. The compound ofclaim 7, or a pharmaceutically acceptable salt thereof, wherein R² andR³ are independently selected from the group consisting of: hydrogen,and C₁-C₃ alkyl.
 9. The compound of claim 8, or a pharmaceuticallyacceptable salt thereof, wherein R² is C₁-C₂ alkyl and R³ is hydrogen.10. The compound of claim 9, or a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable salt thereof wherein R⁵, R⁶,and R⁷ are H, and R⁴ is C₁-C₃-alkyl.
 11. The compound of claim 10, or apharmaceutically acceptable salt thereof, wherein R⁴ is methyl.
 12. Thecompound of claim 11, or a pharmaceutically acceptable salt thereofwherein R¹ is a thieno[3,2-c]pyridinyl or a thieno[2,3-c]pyridinyl,which may be optionally substituted with one to three substituents eachindependently selected from the group consisting of: —OH, C₁-C₃ alkyl,halo, and —O—C₁-C₃ alkyl.
 13. The compound of claim 12, or apharmaceutically acceptable salt thereof wherein R¹ isthieno[3,2-c]pyridin-2-yl or a thieno[2,3-c]pyridin-2-yl, which may beoptionally substituted with one to three substituents each independentlyselected from the group consisting of: —OH, C₁-C₃ alkyl, halo, and—O—C₁-C₃ alkyl.
 14. The compound of claim 13, or a pharmaceuticallyacceptable salt thereof wherein R¹ is a thieno[3,2-c]pyridin-2-yl. 15.The compound of claim 1, wherein said compound is selected from thegroup consisting of:2-[1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;2-[3,4-dimethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;2-[3-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;2-[3-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;2-[4-ethyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;and pharmaceutically acceptable salts thereof.
 16. The compound of claim1, wherein said compound is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof. 17.2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine.18. The compound of claim 13, or a pharmaceutically acceptable saltthereof wherein R¹ is a thieno[2,3-c]pyridin-2-yl.
 19. The compound ofclaim 1, wherein said compound is2-(4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl)thieno[2,3-c]pyridine,or a pharmaceutically acceptable salt thereof.
 20. A method ofinhibiting scar formation, comprising administering to a mammal in needof such treatment a therapeutically effective amount of a compound ofclaim 1, or a pharmaceutically acceptable salt thereof.
 21. The methodof claim 20, where said compound of claim 1 is administered topically.22. The method of claim 20, where said compound is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof.
 23. The use of a2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof, in the manufacture of amedicament for the inhibition of scar formation.
 24. A method oftreating a TGFβ-mediated condition, comprising administering to a mammalin need of such treatment a therapeutically effective amount of acompound of claim 1, or a pharmaceutically acceptable salt thereof. 25.The method of claim 24, wherein said TGFβ-mediated condition is selectedfrom the group comprising: cancer, breast cancer, lung cancer, coloncancer, prostate cancer, ovarian cancer, pancreatic cancer, melanoma,fibrotic diseases, glomerulonephritis, diabetic nephropathy, hepaticfibrosis, pulmonary fibrosis, arterial hyperplasia, restenosis,scleroderma, and dermal scarring.
 26. A pharmaceutical compositioncomprising: a therapeutically effective amount of a compound of claim 1,or pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable excipient.
 27. The pharmaceutical composition of claim 26,wherein said compound is2-[4-methyl-1-(6-methylpyridin-2-yl)-1H-pyrazol-5-yl]thieno[3,2-c]pyridine;or a pharmaceutically acceptable salt thereof.
 28. A topicalpharmaceutical composition comprising: a therapeutically effectiveamount of a compound of claim 1, or pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient suitable fortopical application.